S. Primary microglial cell culture supernatants of LPS and/or JQ
S. Primary microglial cell culture supernatants of LPS and/or JQ1 MS023 web co-treated cells were subjected to ELISA to detect the levels of pro-inflammatory cytokines/chemokines. Therefore, primary microglial cells were treated with 10 ng/mL LPS and/or 500 nM of JQ1 for 2 and 4 h, followed by quantification of Ccl2, Ccl7, and Cxcl10 levels. Values are given in pg/ml. Means and standard deviations of the mean of the three independent experiments are shown (*P value <0.05, **P value <0.001). LPS, lipopolysaccharide; Con., control.pan-BET inhibitor, I-BET, has been proven to protect against LPS-induced endotoxic shock [7]. Another BET inhibitor can disrupt the T-cell-mediated inflammatory response [32]. Among these inhibitors, JQ1 has attracted the most attention because of its significant efficiency in hematological malignancies [33]. Recently, other studies have reported even wider prospective applications for JQ1, such as in attenuating lung fibrosis [34], endotoxemic shock [10], NO synthesis, and innate immunity [35], suggesting that JQ1 may have anti-inflammatory activity. However, none of these studies addressed the effects of JQ1 at the genome-wide expression level in BV-2 microglial cells. We examined BV-2 cell lines as a model of inflammation studies. This is one of the major uses of microglia. Previously, other reports demonstrated that BV-2 cell lines have close resemblance to primary brainmicroglia [36-38]. Since BV-2 cells are easy to culture, they are an important tool to study not only inflammatory processes [38] but also phagocytosis [39]. In the present study, we, for the first time, showed the anti-inflammatory effect of JQ1 on genome-wide mRNA levels in BV-2 microglial cells, a model system for studying inflammation, using RNA-Seq analysis. This study provides the most comprehensive analysis thus far, as the technique provides unbiased profiles, ability to identify novel transcribed regions, compared to microarrays, and can be extremely accurate. This unbiased profiling approach revealed that the importance of BET proteins in the regulation of key inflammatory genes involved in the establishment of innate immunity in BV-2 microglial cells. The results show that the stimulation of BV-2 microglial cells with LPS upregulated numerous inflammatoryJung et al. Journal of Neuroinflammation (2015) 12:Page 15 ofgenes, including Nos2, Il1b, Il1a, Il18, Il1rn, Tnf-, Ptgs2, Nfbiz, Nfbia, Nfb2, Relb, Nfbie, Nfb1, Ifit1, Irf1, Irf7, Irf9, Cxcl10, Ccl4, Ccl7, Ccl2, Ccl3, Ccl12, and Ccl9. Treatment of BV-2 microglial cells with JQ1 resulted in the downregulation of 78 and 118 (P 0.01 and fold change 1.5) of the LPS-inducible genes at 2 and 4 h, respectively, suppressing key LPS-inducible inflammatory genes, including Il1a, Il1b, Nos2, Ptgs2, Irf1, Irf7, Irf9, Ccl2, Ccl7, Ccl9, Ccl12 and Cxcl10 (Figure 5A,B). Il1 is the most widely studied pro-inflammatory gene; the extensively characterized forms of Il1 are Il1a and Il1b [40]. Il1a and Il1b play a crucial role in the development of AD and PD, the pathogenic hallmark of which is CNS inflammation [41,42]. Following CNS damage, Il1 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28212752 is rapidly released from activated microglia, and an elevated level of the Il1 cytokine is an important hallmark of neuroinflammation [43]. In this study, we showed that JQ1 treatment significantly reduced the expression of Il1a and Il1b, which had been increased by LPS stimulation. Thus, the downregulation of Il1a and Il1b through JQ1 could inhibit neuroinflammation as.
