title: Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis.
abstract: BACKGROUND
  We investigated sera from elderly subjects with and without age-related macular degeneration (AMD) for presence of autoantibodies (AAbs) against human macular antigens and characterized their identity.
  
  
  METHODS
  Sera were collected from participants in the Age-Related Maculopathy Ancillary (ARMA) Study, a cross-sectional investigation ancillary to the Health ABC Study, enriched with participants from the general population. The resulting sample (mean age: 79.2±3.9 years old) included subjects with early to advanced AMD (n = 131) and controls (n = 231). Sera were tested by Western blots for immunoreactive bands against human donor macular tissue homogenates. Immunoreactive bands were identified and graded, and odds ratios (OR) calculated. Based on these findings, sera were immunoprecipitated, and subjected to 2D gel electrophoresis (GE). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the targets recognized by circulating AAbs seen on 2D-GE, followed by ELISAs with recombinant proteins to confirm LC-MS/MS results, and quantify autoreactivities.
  
  
  RESULTS
  In AMD, 11 immunoreactive bands were significantly more frequent and 13 were significantly stronger than in controls. Nine of the more frequent bands also showed stronger reactivity. OR estimates ranged between 4.06 and 1.93, and all clearly excluded the null value. Following immunoprecipitation, 2D-GE and LC-MS/MS, five of the possible autoreactivity targets were conclusively identified: two members of the heat shock protein 70 (HSP70) family, HSPA8 and HSPA9; another member of the HSP family, HSPB4, also known as alpha-crystallin A chain (CRYAA); Annexin A5 (ANXA5); and Protein S100-A9, also known as calgranulin B that, when complexed with S100A8, forms calprotectin. ELISA testing with recombinant proteins confirmed, on average, significantly higher reactivities against all targets in AMD samples compared to controls.
  
  
  CONCLUSIONS
  Consistent with other evidence supporting the role of inflammation and the immune system in AMD pathogenesis, AAbs were identified in AMD sera, including early-stage disease. Identified targets may be mechanistically linked to AMD pathogenesis because the identified proteins are implicated in autophagy, immunomodulation, and protection from oxidative stress and apoptosis. In particular, a role in autophagy activation is shared by all five autoantigens, raising the possibility that the detected AAbs may play a role in AMD via autophagy compromise and downstream activation of the inflammasome. Thus, we propose that the detected AAbs provide further insight into AMD pathogenesis and have the potential to contribute to disease biogenesis and progression.
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Iannaccone
authors.affiliation: Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Francesco
authors.initials: F
authors.lastname: Giorgianni
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: David D
authors.initials: DD
authors.lastname: New
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: T J
authors.initials: TJ
authors.lastname: Hollingsworth
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Allison
authors.initials: A
authors.lastname: Umfress
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Albert H
authors.initials: AH
authors.lastname: Alhatem
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.|Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Indira
authors.initials: I
authors.lastname: Neeli
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.|Department of Internal Medicine/Endocrinology, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Nataliya I
authors.initials: NI
authors.lastname: Lenchik
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Jennings
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.|Charles Retina Institute, Memphis, TN, United States of America.
authors.forename: Jorge I
authors.initials: JI
authors.lastname: Calzada
authors.affiliation: Department of Preventive Medicine, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Satterfield
authors.affiliation: Eye Specialty Group, Memphis, TN, United States of America.|Southern College of Optometry, Memphis, TN, United States of America.
authors.forename: Dennis
authors.initials: D
authors.lastname: Mathews
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.|Charles Retina Institute, Memphis, TN, United States of America.
authors.forename: Rocio I
authors.initials: RI
authors.lastname: Diaz
authors.affiliation: National Institute on Aging, NIH, Bethesda, MD, United States of America.
authors.forename: Tamara
authors.initials: T
authors.lastname: Harris
authors.affiliation: Department of Preventive Medicine, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Karen C
authors.initials: KC
authors.lastname: Johnson
authors.affiliation: Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.|Charles Retina Institute, Memphis, TN, United States of America.
authors.forename: Steve
authors.initials: S
authors.lastname: Charles
authors.affiliation: Department of Preventive Medicine, University of Tennessee Health Science Center, Memphis, TN, United States of America.|Sticht Center on Aging, Wake Forest University, Winston-Salem, NC, United States of America.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Kritchevsky
authors.affiliation: Department of Internal Medicine/Endocrinology, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Ivan C
authors.initials: IC
authors.lastname: Gerling
authors.affiliation: Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Sarka
authors.initials: S
authors.lastname: Beranova-Giorgianni
authors.affiliation: Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Marko Z
authors.initials: MZ
authors.lastname: Radic
mesh_terms: D017209:Apoptosis / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood*; D001324:Autoantigens / Q000276:immunology*; D001343:Autophagy / Q000276:immunology*; D015153:Blotting, Western; D002853:Chromatography, Liquid; D016001:Confidence Intervals; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D056747:Immunomodulation; D008268:Macular Degeneration / Q000097:blood* / Q000276:immunology*; D016017:Odds Ratio; D018384:Oxidative Stress / Q000276:immunology*; D053719:Tandem Mass Spectrometry
chemical_list: D001323:Autoantibodies; D001324:Autoantigens