Iannaccone Alessandro, Giorgianni Francesco, New David D, Hollingsworth T J, Umfress Allison, Alhatem Albert H, Neeli Indira, Lenchik Nataliya I, Jennings Barbara J, Calzada Jorge I, Satterfield Suzanne, Mathews Dennis, Diaz Rocio I, Harris Tamara, Johnson Karen C, Charles Steve, Kritchevsky Stephen B, Gerling Ivan C, Beranova-Giorgianni Sarka, Radic Marko Z
Department of Ophthalmology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN, United States of America.
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, United States of America.
PLoS One. 2015 Dec 30;10(12):e0145323. doi: 10.1371/journal.pone.0145323. eCollection 2015.
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.
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.
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.
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.
我们研究了患有和未患有年龄相关性黄斑变性（AMD）的老年受试者血清中针对人黄斑抗原的自身抗体（AAb）的存在情况，并对其特性进行了表征。
从年龄相关性黄斑病变辅助研究（ARMA）的参与者中收集血清，该研究是健康ABC研究的一项横断面辅助调查，招募了来自普通人群的参与者。最终样本（平均年龄：79.2±3.9岁）包括早期至晚期AMD患者（n = 131）和对照组（n = 231）。通过蛋白质免疫印迹法检测血清中针对人供体黄斑组织匀浆的免疫反应条带。对免疫反应条带进行鉴定和分级，并计算比值比（OR）。基于这些发现，对血清进行免疫沉淀，然后进行二维凝胶电泳（2-DE）。液相色谱-串联质谱（LC-MS/MS）用于鉴定二维凝胶电泳上显示出的循环AAb所识别的靶标，随后使用重组蛋白进行酶联免疫吸附测定（ELISA）以确认LC-MS/MS结果并定量自身反应性。
在AMD患者中，11条免疫反应条带的出现频率显著高于对照组，13条免疫反应条带的强度显著高于对照组。9条出现频率较高的条带也表现出更强的反应性。OR估计值在4.06至1.93之间，均明显排除了零值。经过免疫沉淀、二维凝胶电泳和LC-MS/MS分析后，最终确定了5个可能的自身反应性靶标：热休克蛋白70（HSP70）家族的两个成员HSPA8和HSPA9；HSP家族的另一个成员HSPB4，也称为α-晶状体蛋白A链（CRYAA）；膜联蛋白A5（ANXA5）；以及蛋白S100-A9，也称为钙粒蛋白B，它与S,100A8结合时形成钙卫蛋白。使用重组蛋白进行的ELISA检测证实，与对照组相比，AMD样本中针对所有靶标的反应性平均显著更高。
与支持炎症和免疫系统在AMD发病机制中作用的其他证据一致，在AMD血清中鉴定出了自身抗体，包括疾病早期阶段。鉴定出的靶标可能在机制上与AMD发病机制相关，因为所鉴定的蛋白质与自噬、免疫调节以及抗氧化应激和细胞凋亡保护有关。特别是，所有5种自身抗原都具有激活自噬的作用，这增加了检测到的自身抗体可能通过损害自噬和下游激活炎性小体在AMD中发挥作用的可能性。因此，我们认为检测到的自身抗体为AMD发病机制提供了进一步的见解，并有可能促进疾病的发生和发展。
