title: Transcriptome sequencing of microglial cells stimulated with TLR3 and TLR4 ligands.
abstract: BACKGROUND
  Resident macrophages in the CNS microglia become activated and produce proinflammatory molecules upon encountering bacteria or viruses. 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 RNA-seq. 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.
  
  
  RESULTS
  Sequencing assessment and quality evaluation revealed that common and unique patterns of proinflammatory genes were significantly up-regulated in response to TLR3 and TLR4 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) (i.e., IRF1, IRF7, and IRF9) and the epigenetic regulators KDM4A and DNMT3L were significantly up-regulated in both TLR3- and TLR4-stimulated microglia. We also identified 29 previously unidentified genes that are important in immune regulation. In addition, we confirmed the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in TLR3-and TLR4-stimulated primary microglial cells. Moreover, transcriptional start sites (TSSs) and isoforms, as well as differential promoter usage, revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with TLR3 and TLR4. Furthermore, 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Bionanotechnology, Hanyang University, Seoul, 133-791, Republic of Korea. amitabhdas.kn@gmail.com.
authors.forename: Amitabh
authors.initials: A
authors.lastname: Das
authors.affiliation: Department of Molecular & Life Sciences, Hanyang University, Ansan, 426-791, Republic of Korea. jincchai@gmail.com.
authors.forename: Jin Choul
authors.initials: JC
authors.lastname: Chai
authors.affiliation: Department of Molecular & Life Sciences, Hanyang University, Ansan, 426-791, Republic of Korea. eio3492@gmail.com.
authors.forename: Sun Hwa
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Molecular & Life Sciences, Hanyang University, Ansan, 426-791, Republic of Korea. yslee@hanyang.ac.kr.
authors.forename: Young Seek
authors.initials: YS
authors.lastname: Lee
authors.affiliation: Department of Molecular & Life Sciences, Hanyang University, Ansan, 426-791, Republic of Korea. kspark73@gmail.com.
authors.forename: Kyoung Sun
authors.initials: KS
authors.lastname: Park
authors.affiliation: Department of Molecular & Life Sciences, Hanyang University, Ansan, 426-791, Republic of Korea. khjung2@gmail.com.|Institute of Natural Science & Technology, Hanyang University, Ansan, 426-791, South Korea. khjung2@gmail.com.
authors.forename: Kyoung Hwa
authors.initials: KH
authors.lastname: Jung
authors.affiliation: Department of Bionanotechnology, Hanyang University, Seoul, 133-791, Republic of Korea. ygchai@hanyang.ac.kr.|Department of Molecular & Life Sciences, Hanyang University, Ansan, 426-791, Republic of Korea. ygchai@hanyang.ac.kr.
authors.forename: Young Gyu
authors.initials: YG
authors.lastname: Chai
mesh_terms: D000818:Animals; D044127:Epigenesis, Genetic / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D007113:Immunity, Innate / Q000187:drug effects / Q000235:genetics; D018836:Inflammation Mediators / Q000378:metabolism; D050838:Interferon Regulatory Factor-3 / Q000235:genetics; D016899:Interferon-beta / Q000235:genetics; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051196:Toll-Like Receptor 3 / Q000235:genetics*; D051197:Toll-Like Receptor 4 / Q000235:genetics*; D024363:Transcription Initiation Site / Q000187:drug effects / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D059467:Transcriptome / Q000187:drug effects / Q000235:genetics*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
chemical_list: D018836:Inflammation Mediators; D050838:Interferon Regulatory Factor-3; C494233:Irf3 protein, mouse; D008024:Ligands; D008070:Lipopolysaccharides; C519703:TLR3 protein, mouse; C493487:Tlr4 protein, mouse; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D016899:Interferon-beta