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

Abstract

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

HIV-1 gp120 Upregulates Brain-Derived Neurotrophic Factor (BDNF) Expression in BV2 Cells via the Wnt/β-Catenin Signaling Pathway

“…BV2 cell line was originally generated from primary microglial cell cultures (Blasi et al 1990) and has been used frequently as a substitute for primary microglia (Henn et al 2009). A wealth of evidence has revealed that BV2 cells exhibit many similarities with primary microglia (Cai et al 2016; Crotti et al 2014; Das et al 2016; Henn et al 2009; Kim et al 2003). Therefore, the study presented here suggests that human or rat microglia may have some similarity cell responses to HIV-gp120.…”

Interferon Regulatory Factor 5 Mediates Lipopolysaccharide Induced Neuroinflammation

“…These observations indicate that LPSinduced neuroin ammation in BV2 microglia is not mediated by IRF5. Similarly, in a RNA sequencing study, induction of IRF5 expression was only observed in LPS stimulated primary microglia, not BV2 microglia 19,24 . However, amyloid β successfully induced the expression of IRF5 and the subsequent proin ammatory cytokines in BV2 microglia (data not shown), suggesting differential signaling pathways involved in LPS and amyloid β induced activation of BV2 microglia.…”

“…M1 microglia initially respond to the pathological stimulus and promote the destruction of invading pathogens, which is accompanied by the release of pro-in ammatory cytokines, inducing neurotoxicity and acute in ammation 17 . M2 microglia, on the other hand, can produce anti-in ammtory cytokines such as IL-4 and IL-10, which could dampen the pro-in ammatory responses and promote the resolution of in ammation and tissue repair 18,19 . Actually, these pro-and antiin ammatory responses are not "all or none" pattern.…”

Anti-inflammatory role of GM1 and modulatory effects of gangliosides on microglia functions

“…Future studies will address the underlying mechanism(s) and will help identifying novel strategies to lower microglia activation in the context of neuroinflammatory conditions. L-t-PDMP treatment to increase microglial endogenous ganglioside levels recapitulated the effects of exogenous GM1 in BV2 cells, but less so in primary microglia, which are generally more responsive to LPS stimulation than BV2 cells [87,88]. In primary microglia, L-t-PDMP elicited a much weaker anti-inflammatory response compared to the administration of exogenous GM1, decreasing expression of pro-inflammatory genes only at low LPS concentrations, but failing to attenuate microglial inflammatory responses at higher LPS concentrations.…”