title: TLR-independent and P2X7-dependent signaling mediate Alu RNA-induced NLRP3 inflammasome activation in geographic atrophy.
abstract: OBJECTIVE
  Accumulation of Alu RNA transcripts due to DICER1 deficiency in the retinal pigmented epithelium (RPE) promotes geographic atrophy. Recently we showed that Alu RNA activated the NLRP3 inflammasome, leading to RPE cell death via interleukin-18 (IL-18)-mediated MyD88 signaling. However, the molecular basis for NLRP3 inflammasome activation by Alu RNA is not well understood. We sought to decipher the key signaling events triggered by Alu RNA that lead to priming and activation of the NLRP3 inflammasome and, ultimately, to RPE degeneration by investigating the roles of the purinoreceptor P2X7, the transcription factor NF-κB, and the Toll-like receptors (TLRs) in these processes.
  
  
  METHODS
  Human and mouse RPE cells were transfected with a plasmid encoding an Alu element (pAlu) or an in vitro-transcribed Alu RNA. Inflammasome priming was assessed by measuring NLRP3 and IL18 mRNA levels by real-time quantitative PCR. Using immunoblotting, we assessed NF-κB activation by monitoring phosphorylation of its p65 subunit, and inflammasome activation by monitoring caspase-1 cleavage into its active form. RPE degeneration was induced in mice by subretinal transfection of pAlu or Alu RNA. The NF-κB inhibitor BAY 11-7082, the P2X7 receptor antagonist A-740003, and the NLRP3 inflammasome inhibitor glyburide were delivered by intravitreous injections. We studied wild-type (WT) C57Bl/6J, P2rx7(-/-), Nfkb1(-/-), and Tlr23479(-/-) mice. RPE degeneration was assessed by fundus photography and zonula occludens-1 (ZO-1) staining of mouse RPE.
  
  
  RESULTS
  Alu RNA-induced NF-κB activation, independent of TLR-1, -2, -3, -4, -6, -7, and -9 signaling, was required for priming the NLRP3 inflammasome. Nfkb1(-/-) and P2rx7(-/-) mice and WT mice treated with the pharmacological inhibitors of NF-κB, P2X7, or NLRP3, were protected against Alu RNA-induced RPE degeneration.
  
  
  CONCLUSIONS
  NF-κB and P2X7 are critical signaling intermediates in Alu RNA-induced inflammasome priming and RPE degeneration. These molecules are novel targets for rational drug development for geographic atrophy.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Kentucky, Lexington, Kentucky.
authors.forename: Nagaraj
authors.initials: N
authors.lastname: Kerur
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Hirano
authors.forename: Valeria
authors.initials: V
authors.lastname: Tarallo
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Fowler
authors.forename: Ana
authors.initials: A
authors.lastname: Bastos-Carvalho
authors.forename: Tetsuhiro
authors.initials: T
authors.lastname: Yasuma
authors.forename: Reo
authors.initials: R
authors.lastname: Yasuma
authors.forename: Younghee
authors.initials: Y
authors.lastname: Kim
authors.forename: David R
authors.initials: DR
authors.lastname: Hinton
authors.forename: Carsten J
authors.initials: CJ
authors.lastname: Kirschning
authors.forename: Bradley D
authors.initials: BD
authors.lastname: Gelfand
authors.forename: Jayakrishna
authors.initials: J
authors.lastname: Ambati
mesh_terms: D020087:Alu Elements; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D020170:Caspase 1 / Q000378:metabolism; D004195:Disease Models, Animal; D057092:Geographic Atrophy / Q000503:physiopathology*; D005905:Glyburide / Q000494:pharmacology; D006801:Humans; D058847:Inflammasomes / Q000502:physiology*; D020382:Interleukin-18 / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000502:physiology*; D055213:Retinal Pigment Epithelium / Q000166:cytology; D015398:Signal Transduction / Q000502:physiology*; D013450:Sulfones / Q000494:pharmacology; D051193:Toll-Like Receptors / Q000502:physiology*
chemical_list: C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D002352:Carrier Proteins; D058847:Inflammasomes; D020382:Interleukin-18; D016328:NF-kappa B; D009570:Nitriles; D058920:Purinergic P2X Receptor Antagonists; D012333:RNA, Messenger; D058486:Receptors, Purinergic P2X7; D013450:Sulfones; D051193:Toll-Like Receptors; D020170:Caspase 1; D005905:Glyburide
keywords: AMD; NLRP3; inflammasome