Role of Bromodomain and Extra-terminal (BET) Proteins Inhibitor in Lipopolysaccharides (LPS)-induced Microglia Using Transcriptomic and Epigenomic Approaches

Abstract

The immune system includes cells that can recognize TLRs and respond to TLR ligands by expressing several genes that are implicated in innate immunity, which is the first line of mammalian defense against invading pathogens. TLRs are a phylogenetically conserved diverse family of sensors that drive innate immune responses following interactions with PAMPs. TLR3 and TLR4 recognize viral dsRNA Poly (I:C) and bacterial endotoxin LPS, respectively. Importantly, these receptors differ in their downstream adaptor molecules. Thus far, only a few studies have investigated the effects of TLR3 and TLR4 in macrophages. However, a genome-wide search for the effects of these TLRs has not been performed in microglia using global transcriptomic RNA sequencing. Gene expression patterns were determined for the BV-2 microglial cell line when stimulated with viral dsRNA Poly (I:C) or bacterial endotoxin LPS to identify novel transcribed genes, as well as investigate how differences in downstream signaling could influence gene expression in innate immunity. 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-ɑ in macrophages. However, a genome-wide search for JQ1 molecular targets is largely unexplored in microglia or macrophages.

Therefore, we evaluate the anti-inflammatory function and underlying genes targeted by JQ1 in LPS-stimulated BV-2 microglial cells and Bone Marrow Derived Macrophages using two transcriptomic techniques: RNA-seq and quantitative real-time PCR. Associated biological pathways and functional gene ontology were also evaluated.

First, we investigated gene expression patterns for the BV-2 microglial cell line when stimulated with Poly (I:C) or LPS to identify novel transcribed genes, as well as investigate how differences in downstream signaling could influence gene expression in innate immunity. Sequencing assessment and quality evaluation revealed that common and unique patterns of proinflammatory genes were significantly up-regulated in response to TLR3 and TLR4 ligand stimulation. However, the IFN/viral response gene showed a stronger response to TLR3 stimulation than to TLR4 stimulation. Unexpectedly, TLR3 and TLR4 stimulation did not activate IFN-ß and IRF3 in BV-2 microglia. Most importantly, we observed that previously unidentified Transcription Factors (TFs) (Irf1, Irf7, and Irf9), histone demethylases (Kdm4a) and DNA methyltransferases (Dnmt3l) were significantly and selectively expressed in BV-2 microglial cells. We also identified 29 previously unidentified genes that are important in immune regulation. The gene expression levels, transcription start site, isoforms, and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation. Moreover, TF motif analysis (-950 to +50 bp of the 5’ upstream promoters) revealed that the DNA sequences for NF-κB, Irf1, and Stat1 were significantly enriched in TLR3- and TLR4-stimulated microglia. Furthermore, we confirmed the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in LPS treated primary microglial cells. This transcriptomic analysis is the first to show a comparison of the family-wide differential expression of most known immune genes and also reveal transcription evidence of multiple gene families in BV-2 microglial cells. Collectively, these unprecedented findings not only permit a comparison of TLR3- and TLR4-stimulated genes but also identify new genes that have not been previously implicated in innate immunity.

In the second and third part of my thesis we investigated involvement of a novel synthetic BET inhibitor JQ1 on TLR4 stimulated inflammatory genes in microglial cells and BMDMs. Among annotated genes, transcriptional sequencing of microglia and BMDMs treated with JQ1 also revealed selectively reduced expression of cytokines/chemokines, interferon-stimulated genes, and prominent TFs in LPS- stimulated microglia and BMDMs. Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Furthermore, we confirmed 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 different categories related to biological regulation, immune system processes and response to stimuli. These unprecedented results suggest the BET inhibitor JQ1 as a candidate for the prevention or therapeutic treatment of not only inflammation-mediated neurodegenerative diseases but also other inflammatory disorders.

Finally, we also evaluated that in mouse microglia JQ1 regulate target gene expression by inhibiting the genome-wide occurrence of epigenetic enhancer marks (H3K4me1, H3K27ac). Active enhancers are further marked by key TFs PU.1 and BRD4. Along with ‘mimicking’ epigenetic enhancer marks JQ1 also inhibited the binding of key TFs PU.1 and BRD4 responsible for the expression of key inflammatory genes in activated microglia. Our study suggests that genome-wide chromatin modifications, such as H3K4me1, H3K27ac as well as key TFs PU.1 and BRD4 by JQ1 can serve as a new generation inflammatory drugs.

In summary, thesis result indicates that both known and unknown differential expressed genes, TFs, and epigenetic regulators identified in the present study may provide new insight regarding the innate immunity to TLR3 and TLR4 in BV-2 microglial cells. This study also predicts that, while both TLR3 and TLR4 have been evolutionarily selected to induce antiviral gene expression, TLR3 seems to be even more specialized than TLR4 to initiate antiviral responses and that the reverse situation was true for the master TF NF-κB. We also find that novel synthetic BET inhibitor JQ1-dependent molecular targets by ‘mimicking’ genome-wide occurrence of epigenetic enhancer marks as well as key TFs PU.1 and BRD4 on TLR4 stimulated microglial cells. Taken together these findings not only identify family-wide DEGs but also establish a role for BET proteins in mouse microglia or macrophages stimulation and justify the further testing of BET protein-targeting genes in inflammatory diseases.