title: Retinal pigment epithelium cells alter the pro-inflammatory response of retinal microglia to TLR-3 stimulation.
abstract: OBJECTIVE
  Microglia are the local cells of the innate immunity in the retina. Toll-like receptor (TLR) 3 is a receptor of the innate immune system, recognizing viral double-stranded RNA. Retinal pigment epithelium (RPE) cells express TLR-3 and react to TLR-3 stimulation. In this study, we investigated the effect of TLR-3-activated RPE on microglia.
  
  
  METHODS
  Primary porcine RPE cells were prepared from freshly prepared pigs' eyes. Retinal microglia were prepared from porcine retina. Expression of the microglia marker Iba1 was evaluated using immunocytochemistry. RPE cells were treated with polyinosinic/polycytidylic acid (Poly I:C; 100 ng/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) for 24 hr. Either the supernatant was applied to microglia for 6 or 24 hr or microglia cells were directly treated with Poly I:C for 6 and 24 hr. Expression of interleukin (IL)-1ß, IL-6, tumour necrosis factor (TNF)α, IL-10, Cox2 and iNOS was evaluated in quantitative PCR. Phagocytosis was evaluated with a microscope-based and a fluoroscan-based phagocytosis assay.
  
  
  RESULTS
  Retinal pigment epithelium (RPE) cells induce the expression of IL-6, IL-1ß and IL-10 in microglia cells. Microglia cells respond to Poly I:C stimulation in a concentration-dependent manner with the induction of IL-1ß, IL-6, TNFα, Cox2, iNOS and, to a lesser degree, IL-10. Stimulation of microglia cells with supernatant of Poly I:C-treated RPE cells further elevated IL-6, IL-1ß and Cox2 expression, while it reduced the expression of iNOS. No changes in phagocytosis could be detected.
  
  
  CONCLUSIONS
  TLR-3-activated RPE exacerbates inflammatory response of microglia in a differentiated manner. This indicates that viral infections in the RPE may have a proinflammatory influence on retinal microglia.
authors.affiliation: Institute of Ophthalmology, University of Kiel, University Medical Center, Kiel, Germany.
authors.forename: Alexa
authors.initials: A
authors.lastname: Klettner
authors.forename: Timothy
authors.initials: T
authors.lastname: Hamann
authors.forename: Karen
authors.initials: K
authors.lastname: Schlüter
authors.forename: Ralph
authors.initials: R
authors.lastname: Lucius
authors.forename: Johann
authors.initials: J
authors.lastname: Roider
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D007378:Interleukins / Q000235:genetics / Q000378:metabolism; D017628:Microglia / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D010587:Phagocytosis; D011070:Poly I-C / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D055351:Retinal Neurons / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000502:physiology*; D013552:Swine; D051196:Toll-Like Receptor 3 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; D016207:Cytokines; D007378:Interleukins; D051196:Toll-Like Receptor 3; D014409:Tumor Necrosis Factor-alpha; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D011070:Poly I-C
keywords: RPE; TLR-3; inflammatory cytokines; microglia