title: Cocaine-mediated induction of microglial activation involves the ER stress-TLR2 axis.
abstract: BACKGROUND
  Neuroinflammation associated with advanced human immunodeficiency virus (HIV)-1 infection is often exacerbated by chronic cocaine abuse. Cocaine exposure has been demonstrated to mediate up-regulation of inflammatory mediators in in vitro cultures of microglia. The molecular mechanisms involved in this process, however, remain poorly understood. In this study, we sought to explore the underlying signaling pathways involved in cocaine-mediated activation of microglial cells.
  
  
  METHODS
  BV2 microglial cells were exposed to cocaine and assessed for toll-like receptor (TLR2) expression by quantitative polymerase chain reaction (qPCR), western blot, flow cytometry, and immunofluorescence staining. The mRNA and protein levels of cytokines (TNFα, IL-6, MCP-1) were detected by qPCR and ELISA, respectively; level of reactive oxygen species (ROS) production was examined by the Image-iT LIVE Green ROS detection kit; activation of endoplasmic reticulum (ER)-stress pathways were detected by western blot. Chromatin immunoprecipitation (ChIP) assay was employed to discern the binding of activating transcription factor 4 (ATF4) with the TLR2 promoter. Immunoprecipitation followed by western blotting with tyrosine antibody was used to determine phosphorylation of TLR2. Cocaine-mediated up-regulation of TLR2 expression and microglial activation was validated in cocaine-injected mice.
  
  
  RESULTS
  Exposure of microglial cells to cocaine resulted in increased expression of TLR2 with a concomitant induction of microglial activation. Furthermore, this effect was mediated by NADPH oxidase-mediated rapid accumulation of ROS with downstream activation of the ER-stress pathways as evidenced by the fact that cocaine exposure led to up-regulation of pPERK/peIF2α/ATF4 and TLR2. The novel role of ATF4 in the regulation of TLR2 expression was confirmed using genetic and pharmacological approaches.
  
  
  CONCLUSIONS
  xThe current study demonstrates that cocaine-mediated activation of microglia involves up-regulation of TLR2 through the ROS-ER stress-ATF4-TLR2 axis. Understanding the mechanism(s) involved in cocaine-mediated up-regulation of ROS-ER stress/TLR2 expression and microglial activation could have implications for the development of potential therapeutic targets aimed at resolving neuroinflammation in cocaine abusers.
authors: Department of Pharmacology and Experimental Neuroscience, 985880 Nebraska Medical Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA. ke.liao@unmc.edu.
authors: Ke
authors: K
authors: Liao
authors: Department of Pharmacology and Experimental Neuroscience, 985880 Nebraska Medical Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA. minglei.guo@unmc.edu.
authors: Minglei
authors: M
authors: Guo
authors: Department of Pharmacology and Experimental Neuroscience, 985880 Nebraska Medical Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA. fang.niu@unmc.edu.
authors: Fang
authors: F
authors: Niu
authors: Department of Pharmacology and Experimental Neuroscience, 985880 Nebraska Medical Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA. lu.yang@unmc.edu.
authors: Lu
authors: L
authors: Yang
authors: Department of Pharmacology and Experimental Neuroscience, 985880 Nebraska Medical Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA. scallen@unmc.edu.
authors: Shannon E
authors: SE
authors: Callen
authors: Department of Pharmacology and Experimental Neuroscience, 985880 Nebraska Medical Center, University of Nebraska Medical Center, Omaha, NE, 68198, USA. sbuch@unmc.edu.
authors: Shilpa
authors: S
authors: Buch
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D003042:Cocaine / Q000494:pharmacology*; D016207:Cytokines / Q000378:metabolism; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology*; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D008262:Macrophage Activation / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D051195:Toll-Like Receptor 2 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: C491766:Ccl2 protein, rat; D018932:Chemokine CCL2; D016207:Cytokines; D018765:Dopamine Uptake Inhibitors; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D051195:Toll-Like Receptor 2; D014409:Tumor Necrosis Factor-alpha; D003042:Cocaine