title: Antibacterial responses of retinal Müller glia: production of antimicrobial peptides, oxidative burst and phagocytosis.
abstract: BACKGROUND
  We have previously shown that, in response to microbial infection, activated Müller glia secrete inflammatory cytokines/chemokines and exhibit antimicrobial properties. The aim of this study is to understand the mechanisms and the key components involved in this response.
  
  
  METHODS
  Immortalized human retinal Müller glia (MIO-M1 cells) were challenged with Staphylococcus (S) aureus, the leading cause of severe intraocular infection followed by RT2 profile PCR array analysis. The expression of human β-defensin 1 (HBD1), 2 (HBD2), 3 (HBD3), hepcidine and cathelicidin LL37 was checked by RT-PCR and quantified by Taqman qPCR. The expression of AMPs was confirmed at protein level by dot-blot analysis. The production of ROS was measured by dicholoro-dihydro-fluorescein diacetate (DCFH-DA) staining by flow cytometry as well as fluorescence microscopy. The level of nitric oxide (NO) was measured by measuring a stable metabolite, nitrite using the Griess reagent. In vitro killing assay was performed by Live/Dead BacLight staining as well as by dilution plating in suspension and adherent conditions following S. aureus infection. Phagocytosis was measured by CFU enumeration following infection.
  
  
  RESULTS
  PCR array data showed that, in comparison to uninfected control cells, bacterial challenge significantly (> two-fold) induced the expression of 26 genes involved in cytokine/chemokine, antimicrobials, Toll-like receptor, apoptotic, and NF-κB signaling. RT-PCR analysis showed time-dependent increased expression of HBD1, HBD2, HBD3, LL-37, and hepcidin mRNA in bacteria-challenged Müller glia. The expression of these antimicrobial molecules was also increased at the protein level in the culture supernatant, as detected by dot-blot analysis. Additionally, the bacteria-stimulated Müller glia were found to produce reactive oxygen (ROS) and reactive nitrogen (RNS) species. In vitro, killing assays revealed that Müller glia exhibited bactericidal activity against S. aureus in both adherent and suspension cultures. Furthermore, our data demonstrated that Müller glia can phagocytize and kill the bacteria in a time-dependent manner.
  
  
  CONCLUSIONS
  These data suggest that retinal Müller glia behave like classical innate immune cells by producing a variety of antimicrobial molecules in response to bacterial challenge, suggesting their pivotal role in retinal innate defense.
authors.forename: Pawan Kumar
authors.initials: PK
authors.lastname: Singh
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Shiha
authors.affiliation: Department of Ophthalmology/Kresge Eye Institute, Wayne State University School of Medicine, 4717 St, Antoine, Detroit, MI 48201, USA. akuma@med.wayne.edu.
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
mesh_terms: D000704:Analysis of Variance; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000627:therapeutic use; D002461:Cell Line, Transformed; D015169:Colony Count, Microbial; D063928:Ependymoglial Cells / Q000187:drug effects*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects / Q000502:physiology*; D064451:Hepcidins / Q000235:genetics; D006801:Humans; D009573:Nitrites / Q000378:metabolism; D010587:Phagocytosis / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013211:Staphylococcus aureus / Q000502:physiology; D023083:beta-Defensins / Q000235:genetics; D054804:Cathelicidins
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D064451:Hepcidins; D009573:Nitrites; D017382:Reactive Oxygen Species; D023083:beta-Defensins; D054804:Cathelicidins