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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

Top IPA-based canonical pathway analyses at 4 h after LPS stimulation in BV2 cell lines and PM. a, b Ingenuity® Bioinformatics pathway analysis revealed that highly canonical pathways were differentially expressed in BV2 cell lines and PM cells after LPS stimulation. The canonical pathways included in this analysis are shown along the y-axis of the bar chart. The x-axis indicates the statistical significance. Calculated using the right-tailed Fisher exact test, the P value indicates which biologic annotations are significantly associated with the input molecules relative to all functionally characterized mammalian molecules and the yellow threshold line represents the default significance
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Fig3: Top IPA-based canonical pathway analyses at 4 h after LPS stimulation in BV2 cell lines and PM. a, b Ingenuity® Bioinformatics pathway analysis revealed that highly canonical pathways were differentially expressed in BV2 cell lines and PM cells after LPS stimulation. The canonical pathways included in this analysis are shown along the y-axis of the bar chart. The x-axis indicates the statistical significance. Calculated using the right-tailed Fisher exact test, the P value indicates which biologic annotations are significantly associated with the input molecules relative to all functionally characterized mammalian molecules and the yellow threshold line represents the default significance

Mentions: To gain further understanding into the molecular functions of up-regulated genes, we performed IPA (IPA, Ingenuity Systems, http://www.ingenuity.com) [28] to identify the canonical pathways that represent the relevant molecular functions based on functional knowledge inputs. IPA analysis of transcriptome profiling data revealed the highly statistically significant regulation of well-known TLR4-mediated pathways that are important in immune responses, including genes involved in the roles of pattern recognition receptors during the recognition of bacteria and viruses, interferons, and NF-kB signaling, in BV2 cell lines and PM (Fig. 3a, b). Other notable pathways included the TREM1, toll-like receptor, and death receptor signaling pathways. The up-regulation of these functions in response to stimulation with TLR4 stimulation is interesting and indicates that our approach for comparing BV2 cell lines and PM is strong but rather predictable. Interestingly, the pathways that scored as unique in PM relative to microglial cell lines were involved in interferon signaling. The biggest difference between PM and microglial cell lines was therefore a difference in interferon signaling according to IPA analysis. This may reflect the interferon signaling that is known to occur in PM following immune activation.Fig. 3


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)

Top IPA-based canonical pathway analyses at 4 h after LPS stimulation in BV2 cell lines and PM. a, b Ingenuity® Bioinformatics pathway analysis revealed that highly canonical pathways were differentially expressed in BV2 cell lines and PM cells after LPS stimulation. The canonical pathways included in this analysis are shown along the y-axis of the bar chart. The x-axis indicates the statistical significance. Calculated using the right-tailed Fisher exact test, the P value indicates which biologic annotations are significantly associated with the input molecules relative to all functionally characterized mammalian molecules and the yellow threshold line represents the default significance
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Top IPA-based canonical pathway analyses at 4 h after LPS stimulation in BV2 cell lines and PM. a, b Ingenuity® Bioinformatics pathway analysis revealed that highly canonical pathways were differentially expressed in BV2 cell lines and PM cells after LPS stimulation. The canonical pathways included in this analysis are shown along the y-axis of the bar chart. The x-axis indicates the statistical significance. Calculated using the right-tailed Fisher exact test, the P value indicates which biologic annotations are significantly associated with the input molecules relative to all functionally characterized mammalian molecules and the yellow threshold line represents the default significance
Mentions: To gain further understanding into the molecular functions of up-regulated genes, we performed IPA (IPA, Ingenuity Systems, http://www.ingenuity.com) [28] to identify the canonical pathways that represent the relevant molecular functions based on functional knowledge inputs. IPA analysis of transcriptome profiling data revealed the highly statistically significant regulation of well-known TLR4-mediated pathways that are important in immune responses, including genes involved in the roles of pattern recognition receptors during the recognition of bacteria and viruses, interferons, and NF-kB signaling, in BV2 cell lines and PM (Fig. 3a, b). Other notable pathways included the TREM1, toll-like receptor, and death receptor signaling pathways. The up-regulation of these functions in response to stimulation with TLR4 stimulation is interesting and indicates that our approach for comparing BV2 cell lines and PM is strong but rather predictable. Interestingly, the pathways that scored as unique in PM relative to microglial cell lines were involved in interferon signaling. The biggest difference between PM and microglial cell lines was therefore a difference in interferon signaling according to IPA analysis. This may reflect the interferon signaling that is known to occur in PM following immune activation.Fig. 3

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