Tj Hollingsworth

Circulating Autoantibodies in Age-Related Macular Degeneration Recognize Human Macular Tissue Antigens Implicated in Autophagy, Immunomodulation, and Protection from Oxidative Stress and Apoptosis.

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.

Retinal pigment epithelium and microglia express the CD5 antigen-like protein, a novel autoantigen in age-related macular degeneration.

Abstract We report on a novel autoantigen expressed in human macular tissues, identified following an initial Western blot (WB)‐based screening of sera from subjects with age‐related macular degeneration (AMD) for circulating auto‐antibodies (AAbs) recognizing macular antigens. Immunoprecipitation, 2D‐gel electrophoresis (2D‐GE) and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS), direct enzyme‐linked immunosorbent assays (ELISA), WBs, immunohistochemistry (IHC), human primary and ARPE‐19 immortalized cell cultures were used to characterize this novel antigen. An approximately 40‐kDa autoantigen in AMD was identified as the scavenger receptor CD5 antigen‐like protein (CD5L), also known as apoptosis inhibitor of macrophage (AIM). CD5L/AIM was localized to human RPE by IHC and WB methods and to retinal microglial cells by IHC. ELISAs with recombinant CD5L/AIM on a subset of AMD sera showed a nearly 2‐fold higher anti‐CD5L/AIM reactivity in AMD vs. Control sera (p = 0.000007). Reactivity ≥0.4 was associated with 18‐fold higher odds of having AMD (χ2 = 21.42, p = 0.00063). Circulating CD5L/AIM levels were also nearly 2‐fold higher in AMD sera compared to controls (p = 0.0052). The discovery of CD5L/AIM expression in the RPE and in retinal microglial cells adds to the known immunomodulatory roles of these cells in the retina. The discovery of AAbs recognizing CD5L/AIM identifies a possible novel disease biomarker and suggest a potential role for CD5L/AIM in the pathogenesis of AMD in situ. The possible mechanisms via which anti‐CD5L/AIM AAbs may contribute to AMD pathogenesis are discussed. In particular, since CD5L is known to stimulate autophagy and to participate in oxidized LDL uptake in macrophages, we propose that anti‐CD5L/AIM auto‐antibodies may play a role in drusen biogenesis and inflammatory RPE damage in AMD. Graphical abstract Sera of subjects with age‐related macular degeneration exhibit auto‐antibodies directed against an antigen that, following 2D gel and mass spectrometry experiments, was discovered to be the CD5‐like (CD5L) protein, a secreted scavenger receptor known to be produced by macrophages. Immunohistochemical experiments reveal that this antigen is expressed also throughout the cytoplasm and, more discretely, at the nuclear level (inset) by human retinal pigment epithelium cell lines. Confocal immunohistochemical microscopy experiments show also that CD5L reactivity in human macular neuroretinal tissue section colocalizes with reactivity for Iba1, a known microglial specific marker. Figure. No Caption available. HighlightsHuman retinal pigment epithelium and retinal microglia express the CD5 antigen‐like protein (CD5L).AMD patients exhibit autoantibodies against CD5L.Anti‐CD5L ELISA reactivity ≥0.4 is associated with 18‐fold higher odds of AMD.Anti‐CD5L autoreactivity could be a potential novel AMD biomarker with possible pathogenic potential.

Murine Retinal Citrullination Declines With Age and is Mainly Dependent on Peptidyl Arginine Deiminase 4 (PAD4)

Purpose Citrullination is a post-translational modification (PTM) that serves many normal physiological functions. Studies have shown that this PTM—along with expression of the catalyzing enzymes, peptidyl arginine deiminases (PADs)—are increased in autoimmune and age-related pathologies. PAD2 retinal expression has been previously documented in rat and human. Herein, we report on the expression levels and patterns of PAD2, PAD4, and retinal citrullination in the murine retina with age. Methods Wild-type (WT) and Pad4-/- (PAD4KO) mice ages 0.5, 0.75, 1, 3, 6, and 9 months were investigated after euthanasia and eye enucleation. Retinal lysates from 3-month-old mice were probed for PAD4 by western blot. Whole eyes were fixed, cryosectioned, and probed using an anti-PAD2/4 antibody (Ab), a specific anti-PAD4 Ab, and F95 anti-citrullinated peptide Ab. Fluorescent intensities were quantified with ImageJ. Results WT retinas show different levels of PAD4 expression in distinct retinal layers, with the most intense labeling in inner retinal layers, while PAD4KO mice lacked retinal PAD4. Using a nonspecific anti-PAD2/4 Ab, PAD reactivity observed in PAD4KO mice was attributed to PAD2. In WT, both PAD2 and PAD4 expression levels decrease significantly with age while low-level residual PAD2 expression was seen in PAD4KO mice. Citrullination levels in WT retinas paralleled PAD4 expression, with PAD4KO mice exhibiting consistently minimal citrullination. Conclusions Both PAD2 and PAD4 expression and citrullination decrease with age in the murine retina. However, in the absence of PAD4, retinal citrullination is nearly abolished, indicating that PAD4 is a main effector for retinal citrullination under physiological conditions.