title: P2Y1 Receptor Signaling Contributes to High Salt-Induced Priming of the NLRP3 Inflammasome in Retinal Pigment Epithelial Cells.
abstract: BACKGROUND
  Systemic hypertension is a risk factor of age-related macular degeneration (AMD), a chronic inflammatory disease. Acute hypertension is caused by increased extracellular osmolarity after intake of dietary salt (NaCl). We determined in cultured human retinal pigment epithelial (RPE) cells whether high extracellular NaCl alters the gene expression of inflammasome-associated proteins, and whether autocrine/paracrine purinergic (P2) receptor signaling contributes to the NaCl-induced NLRP3 gene expression.
  
  
  RESULTS
  Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Gene and protein expression levels were determined with real-time RT-PCR and Western blot analysis, respectively. IL-1β and IL-18 levels were evaluated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. High extracellular NaCl induced NLRP3 and pro-IL-1β gene expression, while the gene expression of further inflammasome-associated proteins (NLRP1, NLRP2, NLRP6, NLRP7, NLRP12, NLRC4, AIM2, ASC, procaspase-1, pro-IL-18) was not altered or below the detection threshold. The NaCl-induced NLRP3 gene expression was partially dependent on the activities of phospholipase C, IP3 receptors, protein kinase C, the serum and glucocorticoid-regulated kinase, p38 MAPK, ERK1/2, JNK, PI3K, and the transcription factors HIF-1 and NFAT5. Pannexin-dependent ATP release and P2Y1 receptor activation is required for the full induction of NLRP3 gene expression. High NaCl induced a transient increase of the NLRP3 protein level and a moderate NLRP3 inflammasome activation, as indicated by the transient increase of the cytosolic level of mature IL-1β. High NaCl also induced secretion of IL-18.
  
  
  CONCLUSIONS
  High extracellular NaCl induces priming of the NLRP3 inflammasome in RPE cells, in part via P2Y1 receptor signaling. The inflammasome priming effect of NaCl suggests that high intake of dietary salt may promote local retinal inflammation implicated in the development of AMD.
authors: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors: Philipp
authors: P
authors: Prager
authors: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors: Margrit
authors: M
authors: Hollborn
authors: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors: Anja
authors: A
authors: Steffen
authors: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors: Peter
authors: P
authors: Wiedemann
authors: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.|Helios Klinikum Aue, Aue, Germany.
authors: Leon
authors: L
authors: Kohen
authors: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors: Andreas
authors: A
authors: Bringmann
mesh_terms: D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein / Q000235:genetics / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1 / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction; D012965:Sodium Chloride / Q000008:administration & dosage*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D058847:Inflammasomes; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D058472:Receptors, Purinergic P2Y1; D014157:Transcription Factors; D012965:Sodium Chloride