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RNA sequencing reveals distinct mechanisms underlying BET inhibitor JQ1-mediated modulation of the LPS-induced activation of BV-2 microglial cells.

Jung KH, Das A, Chai JC, Kim SH, Morya N, Park KS, Lee YS, Chai YG - J Neuroinflammation (2015)

Bottom Line: With a cutoff value of P ≤ 0.01 and fold change ≥1.5 log2, the expression level of 214 and 301 genes, including pro-inflammatory cytokine, chemokine, and transcription factors, was found to be upregulated in BV-2 cells stimulated with LPS for 2 and 4 h, respectively.Among these annotated genes, we found that JQ1 selectively reduced the expression of 78 and 118 genes (P ≤ 0.01, and fold change ≥ 1.5, respectively).Furthermore, we confirmed that JQ1 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines in the supernatants of LPS-treated primary microglial cells isolated from 3-day-old ICR mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan, Gyeonggi-do, 426-791, South Korea. khjung2@gmail.com.

ABSTRACT

Background: Microglial cells become rapidly activated through interaction with pathogens, and their persistent activation is associated with the production and secretion of various pro-inflammatory genes, cytokines, and chemokines, which may initiate or amplify neurodegenerative diseases. Bromodomain and extraterminal domain (BET) proteins are a group of epigenetic regulators that associate with acetylated histones and facilitate the transcription of target genes. A novel synthetic BET inhibitor, JQ1, was proven to exert immunosuppressive activities by inhibiting the expression of IL-6 and Tnf-α in macrophages. However, a genome-wide search for JQ1 molecular targets is largely unexplored in microglia.

Methods: The present study was aimed at evaluating the anti-inflammatory function and underlying genes targeted by JQ1 in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells using two transcriptomic techniques: global transcriptomic biological duplicate RNA sequencing and quantitative real-time PCR. Associated biological pathways and functional gene ontology were also evaluated.

Results: With a cutoff value of P ≤ 0.01 and fold change ≥1.5 log2, the expression level of 214 and 301 genes, including pro-inflammatory cytokine, chemokine, and transcription factors, was found to be upregulated in BV-2 cells stimulated with LPS for 2 and 4 h, respectively. Among these annotated genes, we found that JQ1 selectively reduced the expression of 78 and 118 genes (P ≤ 0.01, and fold change ≥ 1.5, respectively). Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Furthermore, we confirmed that JQ1 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines in the supernatants of LPS-treated primary microglial cells isolated from 3-day-old ICR mice. Utilizing functional group analysis, the genes affected by JQ1 were classified into four categories related to biological regulation, immune system processes, and response to stimuli. Moreover, the biological pathways and functional genomics obtained in this study may facilitate the suppression of different key inflammatory genes through JQ1-treated BV-2 microglial cells.

Conclusions: These unprecedented results suggest the BET inhibitor JQ1 as a candidate for the prevention or therapeutic treatment of inflammation-mediated neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus

JQ1 suppresses a specific subset of LPS-inducible genes. (A) Heat map representation of the top 50 expression levels of genes that were downregulated (P ≤ 0.01 and fold change ≥1.5) by JQ1 at 2 h (left panel) and (B) 4 h (right panel) after LPS stimulation of two independent BV-2 microglial cultures. Biological replicates (n = 2) for each condition were combined separately, and the heat map were generated with the Multi Experiment Viewer (version 4.8) software. (C and D) UCSC Browser images representing the normalized RNA-Seq read density in JQ1-downregulated inflammatory genes at 2 and 4 h in LPS-stimulated BV-2 microglial cells compared to the control, respectively. LPS, lipopolysaccharide.
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Fig5: JQ1 suppresses a specific subset of LPS-inducible genes. (A) Heat map representation of the top 50 expression levels of genes that were downregulated (P ≤ 0.01 and fold change ≥1.5) by JQ1 at 2 h (left panel) and (B) 4 h (right panel) after LPS stimulation of two independent BV-2 microglial cultures. Biological replicates (n = 2) for each condition were combined separately, and the heat map were generated with the Multi Experiment Viewer (version 4.8) software. (C and D) UCSC Browser images representing the normalized RNA-Seq read density in JQ1-downregulated inflammatory genes at 2 and 4 h in LPS-stimulated BV-2 microglial cells compared to the control, respectively. LPS, lipopolysaccharide.

Mentions: To investigate whether the broad-spectrum BET protein inhibitor JQ1 is also a broad-spectrum, anti-inflammatory agent, we tested its efficacy as an immunomodulatory drug that could counter microglia-mediated inflammation. We first examined whether JQ1 could alter the expression of inflammation-related genes in microglial cells. The effect of JQ1 on LPS-induced inflammatory genes was examined at 2 and 4 h of LPS stimulation. Most of the genes were significantly suppressed by JQ1 in a dose-dependent manner (Figure 4); indeed, we observed that 500-nM JQ1, a dose used in previous publications [9,27,28], led to a marked reduction of inflammatory gene expression in BV-2 microglial cells. Furthermore, we exposed BV-2 microglial cells to LPS and treated them biologically inactive enantiomer JQ1 (−). As expected, expression levels of inflammatory genes were not reduced in JQ1 (−)-treated BV-2 microglial cells (Additional file 3: Figure S2). We then exposed the BV-2 microglial cells to LPS and concomitantly treated them with JQ1 (+) for both time periods and compared the gene expression profile from the group treated with LPS alone with that obtained from the group treated with JQ1 + LPS. Treatment of BV-2 microglial cells with JQ1 and LPS resulted in the downregulation (P ≤ 0.01, and fold change ≥1.5) of 78 and 118 of the LPS-inducible genes at 2 and 4 h, respectively (Figure 5). JQ1 suppressed the expression of key LPS-inducible inflammation- and immunity-related genes, including Il1a, Il1b, Irg1, Ptgs2, iNOS, Ccl2, Ccl4, Ccl7, Ccl12, Cxcl10, Irf1, Irf7, and Irf9 (Figure 5A-D). Most interestingly, an inhibitor of the NF-κB transcription factor, Nfkbia, was not suppressed by JQ1. A crucial inflammatory gene, Tnf-α (Additional file 4: Figure S3), as well as other inflammation- and immunity-related genes, such as Saa3, Nfkbiz, Tnfaip2, Nfκb2, and Ccl3, were unaffected by JQ1, suggesting that JQ1-treated, LPS-inducible gene expression is highly selective. Consistent with our findings, Nicodeme et al. [7] reported that significant inflammatory genes Tnf-α, Ccl3 were unaffected by another synthetic BET family proteins (I-BET) in bone marrow-derived macrophages (BMDM). They observed that following I-BET treatment higher BET levels at Tnf-α locus were associated with largely unchanged levels of positive transcriptional elongation factor b, RNA polymerase II, and RNA polymerase II S2. In contrast, Belkina et al. [10] demonstrated that JQ1 is a potent inhibitor of Tnf-α production in BMDM. This mechanism is the subject of ongoing investigations. This is an exciting area that we are keenly pursuing further.Figure 4


RNA sequencing reveals distinct mechanisms underlying BET inhibitor JQ1-mediated modulation of the LPS-induced activation of BV-2 microglial cells.

Jung KH, Das A, Chai JC, Kim SH, Morya N, Park KS, Lee YS, Chai YG - J Neuroinflammation (2015)

JQ1 suppresses a specific subset of LPS-inducible genes. (A) Heat map representation of the top 50 expression levels of genes that were downregulated (P ≤ 0.01 and fold change ≥1.5) by JQ1 at 2 h (left panel) and (B) 4 h (right panel) after LPS stimulation of two independent BV-2 microglial cultures. Biological replicates (n = 2) for each condition were combined separately, and the heat map were generated with the Multi Experiment Viewer (version 4.8) software. (C and D) UCSC Browser images representing the normalized RNA-Seq read density in JQ1-downregulated inflammatory genes at 2 and 4 h in LPS-stimulated BV-2 microglial cells compared to the control, respectively. LPS, lipopolysaccharide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: JQ1 suppresses a specific subset of LPS-inducible genes. (A) Heat map representation of the top 50 expression levels of genes that were downregulated (P ≤ 0.01 and fold change ≥1.5) by JQ1 at 2 h (left panel) and (B) 4 h (right panel) after LPS stimulation of two independent BV-2 microglial cultures. Biological replicates (n = 2) for each condition were combined separately, and the heat map were generated with the Multi Experiment Viewer (version 4.8) software. (C and D) UCSC Browser images representing the normalized RNA-Seq read density in JQ1-downregulated inflammatory genes at 2 and 4 h in LPS-stimulated BV-2 microglial cells compared to the control, respectively. LPS, lipopolysaccharide.
Mentions: To investigate whether the broad-spectrum BET protein inhibitor JQ1 is also a broad-spectrum, anti-inflammatory agent, we tested its efficacy as an immunomodulatory drug that could counter microglia-mediated inflammation. We first examined whether JQ1 could alter the expression of inflammation-related genes in microglial cells. The effect of JQ1 on LPS-induced inflammatory genes was examined at 2 and 4 h of LPS stimulation. Most of the genes were significantly suppressed by JQ1 in a dose-dependent manner (Figure 4); indeed, we observed that 500-nM JQ1, a dose used in previous publications [9,27,28], led to a marked reduction of inflammatory gene expression in BV-2 microglial cells. Furthermore, we exposed BV-2 microglial cells to LPS and treated them biologically inactive enantiomer JQ1 (−). As expected, expression levels of inflammatory genes were not reduced in JQ1 (−)-treated BV-2 microglial cells (Additional file 3: Figure S2). We then exposed the BV-2 microglial cells to LPS and concomitantly treated them with JQ1 (+) for both time periods and compared the gene expression profile from the group treated with LPS alone with that obtained from the group treated with JQ1 + LPS. Treatment of BV-2 microglial cells with JQ1 and LPS resulted in the downregulation (P ≤ 0.01, and fold change ≥1.5) of 78 and 118 of the LPS-inducible genes at 2 and 4 h, respectively (Figure 5). JQ1 suppressed the expression of key LPS-inducible inflammation- and immunity-related genes, including Il1a, Il1b, Irg1, Ptgs2, iNOS, Ccl2, Ccl4, Ccl7, Ccl12, Cxcl10, Irf1, Irf7, and Irf9 (Figure 5A-D). Most interestingly, an inhibitor of the NF-κB transcription factor, Nfkbia, was not suppressed by JQ1. A crucial inflammatory gene, Tnf-α (Additional file 4: Figure S3), as well as other inflammation- and immunity-related genes, such as Saa3, Nfkbiz, Tnfaip2, Nfκb2, and Ccl3, were unaffected by JQ1, suggesting that JQ1-treated, LPS-inducible gene expression is highly selective. Consistent with our findings, Nicodeme et al. [7] reported that significant inflammatory genes Tnf-α, Ccl3 were unaffected by another synthetic BET family proteins (I-BET) in bone marrow-derived macrophages (BMDM). They observed that following I-BET treatment higher BET levels at Tnf-α locus were associated with largely unchanged levels of positive transcriptional elongation factor b, RNA polymerase II, and RNA polymerase II S2. In contrast, Belkina et al. [10] demonstrated that JQ1 is a potent inhibitor of Tnf-α production in BMDM. This mechanism is the subject of ongoing investigations. This is an exciting area that we are keenly pursuing further.Figure 4

Bottom Line: With a cutoff value of P ≤ 0.01 and fold change ≥1.5 log2, the expression level of 214 and 301 genes, including pro-inflammatory cytokine, chemokine, and transcription factors, was found to be upregulated in BV-2 cells stimulated with LPS for 2 and 4 h, respectively.Among these annotated genes, we found that JQ1 selectively reduced the expression of 78 and 118 genes (P ≤ 0.01, and fold change ≥ 1.5, respectively).Furthermore, we confirmed that JQ1 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines in the supernatants of LPS-treated primary microglial cells isolated from 3-day-old ICR mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan, Gyeonggi-do, 426-791, South Korea. khjung2@gmail.com.

ABSTRACT

Background: Microglial cells become rapidly activated through interaction with pathogens, and their persistent activation is associated with the production and secretion of various pro-inflammatory genes, cytokines, and chemokines, which may initiate or amplify neurodegenerative diseases. Bromodomain and extraterminal domain (BET) proteins are a group of epigenetic regulators that associate with acetylated histones and facilitate the transcription of target genes. A novel synthetic BET inhibitor, JQ1, was proven to exert immunosuppressive activities by inhibiting the expression of IL-6 and Tnf-α in macrophages. However, a genome-wide search for JQ1 molecular targets is largely unexplored in microglia.

Methods: The present study was aimed at evaluating the anti-inflammatory function and underlying genes targeted by JQ1 in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells using two transcriptomic techniques: global transcriptomic biological duplicate RNA sequencing and quantitative real-time PCR. Associated biological pathways and functional gene ontology were also evaluated.

Results: With a cutoff value of P ≤ 0.01 and fold change ≥1.5 log2, the expression level of 214 and 301 genes, including pro-inflammatory cytokine, chemokine, and transcription factors, was found to be upregulated in BV-2 cells stimulated with LPS for 2 and 4 h, respectively. Among these annotated genes, we found that JQ1 selectively reduced the expression of 78 and 118 genes (P ≤ 0.01, and fold change ≥ 1.5, respectively). Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Furthermore, we confirmed that JQ1 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines in the supernatants of LPS-treated primary microglial cells isolated from 3-day-old ICR mice. Utilizing functional group analysis, the genes affected by JQ1 were classified into four categories related to biological regulation, immune system processes, and response to stimuli. Moreover, the biological pathways and functional genomics obtained in this study may facilitate the suppression of different key inflammatory genes through JQ1-treated BV-2 microglial cells.

Conclusions: These unprecedented results suggest the BET inhibitor JQ1 as a candidate for the prevention or therapeutic treatment of inflammation-mediated neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus