Deciphering the oxidation status of biomolecules in synovial fluids and tissues acting as damage associated molecular patterns (DAMPs) in rheumatoid arthritis

关节炎可由退行性变或自身免疫引起，是工业化国家致残和提前退休的主要原因。例如，超过200万德国公民被诊断为软骨退化和/或一个或多个关节功能丧失的明显影响。因此，保健的社会经济后果和费用是巨大的。更糟糕的是，这种疾病的发病率在不断增加。活性氧（ROS）的过量产生，如次合唱酸（HOCl）和羟基自由基，是炎症性疾病的重要标志，尤其是类风湿关节炎（RA）。这些高活性物质具有修饰几乎所有生物分子的潜力：对蛋白质、糖胺聚糖（软骨细胞外基质的主要碳水化合物）和磷脂中的（多）不饱和脂肪酸酰基残基的影响可以明确证实。然而，对于氧化修饰的脂类（具有活性醛）与丰富的蛋白质或糖胺聚糖的氨基反应形成的产物，我们的知识相当缺乏。此外，越来越多的人认识到，由ROS修饰的内源性分子（损伤相关分子模式；DAMPs）激活细胞受体，并伴有炎症增加。我们假设这些已知但“未研究”的物质与炎症性疾病的发病机制特别相关，即氧化化合物与炎症性风湿病的发病机制特别相关，因此具有诊断和治疗重要性。我们在不同的复杂程度上处理这些方面。首先，我们将合成氧化和氯化脂肪酸和磷脂。通过高分辨率色谱、质谱和核磁共振光谱对这些产品进行详细表征是一项非常具有挑战性的任务，因为方法必须首先适应这些分子。更具挑战性的将是分析氧化脂肪酸/脂质与糖胺聚糖或肽之间形成的反应产物，这些反应产物模拟了与风湿病有关的蛋白质（润滑蛋白、聚集蛋白）的活性位点。其次，在第一个目标中确定的氧化分子类别将在风湿性疾病患者的滑膜液和滑膜提取物中进行研究，并与疾病的严重程度相关。第三，我们将判断已鉴定的氧化PL、GAG和作为氧化DAMPs的蛋白质在细胞免疫反应中的相关性。为了完成这项任务，将在体外通过测量典型的炎症标志物（如干扰素- γ和由细胞释放的选定细胞因子）来研究它们对T细胞、抗原提呈细胞和类风湿滑膜单核细胞的影响。

Arthritis, which can be due to degeneration or autoimmunity, is a major cause of disability and early retirement in the industrialized countries. For instance, more than 2 million German citizens were diagnosed to be markedly affected by cartilage degradation and/or loss of function of one or more joints. Thus, the socio-economic consequences and costs for health care are immense. Even worse, the incidence of the disease is continuously increasing.The excessive production of reactive oxygen species (ROS), such as hypochorus acid (HOCl) and hydroxyl radicals, is an important hallmark of inflammatory diseases, especially for rheumatoid arthritis (RA). These highly reactive species have the potential to modify nearly all biomolecules: effects towards proteins, glycosaminoglycans (the prime carbohydrates of the extracellular matrix of cartilage) and the (poly)unsaturated fatty acyl residues within phospholipids could be unequivocally substantiated. However, there is a considerable lack of knowledge about the products that are formed, for example, by oxidatively modified lipids (with reactive aldehyde groups) reacting with the amino groups of abundant proteins or glycosaminoglycans. Additionally, it is increasingly recognized that endogenous molecules modified by ROS (damage-associated molecular patterns; DAMPs) activate cellular receptors that are accompanied by increased inflammation. We hypothesize that these known but "non-studied" substances are particularly relevant for the pathogenesis of inflammatory diseases, i.e. that oxidized compounds are particularly relevant for the pathogenesis of inflammatory rheumatic diseases and are thus of diagnostic and therapeutic importance. We deal with these aspects on different levels of complexity. First, we will synthesize oxidized and chlorinated fatty acids and phospholipids. The detailed characterization of these products by high resolution chromatography, mass spectrometry and NMR spectroscopy is a very challenging task, as methods have to be first adapted to these molecules. Even more challenging will be the analysis of reaction products formed between oxidized fatty acids/lipids and glycosaminoglycans or peptides mimicking the active sites of proteins (lubricin, aggrecan) involved in rheumatic diseases. Second, oxidized molecule classes identified in the first aim will be investigated both in synovial fluid and extracts from the synovial membranes from patients suffering from rheumatic diseases in relation to the severity of the disease. Third, we will judge the relevance of identified oxidized PL, GAG, and proteins as oxidative DAMPs on the cellular immune response. For this task, their effects on T cells, antigen presenting cells and monocytes from the rheumatoid synovium will be studied in vitro by measuring typical inflammation markers such as interferon-gamma and selected cytokines that are released by the cells.