title: Minocycline counter-regulates pro-inflammatory microglia responses in the retina and protects from degeneration.
abstract: BACKGROUND
  Microglia reactivity is a hallmark of retinal degenerations and overwhelming microglial responses contribute to photoreceptor death. Minocycline, a semi-synthetic tetracycline analog, has potent anti-inflammatory and neuroprotective effects. Here, we investigated how minocycline affects microglia in vitro and studied its immuno-modulatory properties in a mouse model of acute retinal degeneration using bright white light exposure.
  
  
  METHODS
  LPS-treated BV-2 microglia were stimulated with 50 μg/ml minocycline for 6 or 24 h, respectively. Pro-inflammatory gene transcription was determined by real-time RT-PCR and nitric oxide (NO) secretion was assessed using the Griess reagent. Caspase 3/7 levels were determined in 661W photoreceptors cultured with microglia-conditioned medium in the absence or presence of minocycline supplementation. BALB/cJ mice received daily intraperitoneal injections of 45 mg/kg minocycline, starting 1 day before exposure to 15.000 lux white light for 1 hour. The effect of minocycline treatment on microglial reactivity was analyzed by immunohistochemical stainings of retinal sections and flat-mounts, and messenger RNA (mRNA) expression of microglia markers was determined using real-time RT-PCR and RNA-sequencing. Optical coherence tomography (OCT) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings were used to measure the extent of retinal degeneration and photoreceptor apoptosis.
  
  
  RESULTS
  Stimulation of LPS-activated BV-2 microglia with minocycline significantly diminished the transcription of the pro-inflammatory markers CCL2, IL6, and inducible nitric oxide synthase (iNOS). Minocycline also reduced the production of NO and dampened microglial neurotoxicity on 661W photoreceptors. Furthermore, minocycline had direct protective effects on 661W photoreceptors by decreasing caspase 3/7 activity. In mice challenged with white light, injections of minocycline strongly decreased the number of amoeboid alerted microglia in the outer retina and down-regulated the expression of the microglial activation marker translocator protein (18 kDa) (TSPO), CD68, and activated microglia/macrophage whey acidic protein (AMWAP) already 1 day after light exposure. Furthermore, RNA-seq analyses revealed the potential of minocycline to globally counter-regulate pro-inflammatory gene transcription in the light-damaged retina. The severe thinning of the outer retina and the strong induction of photoreceptor apoptosis induced by light challenge were nearly completely prevented by minocycline treatment as indicated by a preserved retinal structure and a low number of apoptotic cells.
  
  
  CONCLUSIONS
  Minocycline potently counter-regulates microgliosis and light-induced retinal damage, indicating a promising concept for the treatment of retinal pathologies.
authors.affiliation: Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, 50931, Cologne, Germany. rebecca.scholz@uk-koeln.de.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Scholz
authors.affiliation: Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, 50931, Cologne, Germany. m.sobotka@gmx.de.
authors.forename: Markus
authors.initials: M
authors.lastname: Sobotka
authors.affiliation: Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, 50931, Cologne, Germany. albert.caramoy@uk-koeln.de.
authors.forename: Albert
authors.initials: A
authors.lastname: Caramoy
authors.affiliation: Center of Excellence for Fluorescent Bioanalytics, University of Regensburg, 93053, Regensburg, Germany. thomas.stempfl@kfb-regensburg.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Stempfl
authors.affiliation: Center of Excellence for Fluorescent Bioanalytics, University of Regensburg, 93053, Regensburg, Germany. christoph.moehle@kfb-regensburg.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Moehle
authors.affiliation: Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, 50931, Cologne, Germany. thomas.langmann@uk-koeln.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Langmann
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D018836:Inflammation Mediators / Q000378:metabolism; D008027:Light / Q000009:adverse effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D017628:Microglia / Q000187:drug effects* / Q000473:pathology*; D008911:Minocycline / Q000494:pharmacology*; D009410:Nerve Degeneration / Q000517:prevention & control; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism; D012160:Retina / Q000473:pathology*; D012162:Retinal Degeneration / Q000188:drug therapy* / Q000473:pathology; D012164:Retinal Diseases / Q000188:drug therapy / Q000473:pathology
chemical_list: D000893:Anti-Inflammatory Agents; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D018696:Neuroprotective Agents; D009569:Nitric Oxide; D020169:Caspases; D008911:Minocycline