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

关节炎，这可能是由于退化或自身免疫性，是一个主要的原因，残疾和提前退休的工业化国家。例如，超过200万德国公民被诊断出明显受到软骨退化和/或一个或多个关节功能丧失的影响。因此，社会经济后果和保健费用是巨大的。活性氧自由基（reactive oxygen species，ROS）的过度产生，如羟自由基（hydroxyl radicals，HOCl）和次氯酸（hypochorus acid，HOCl），是炎症性疾病的重要标志，尤其是类风湿关节炎（rheumatoid arthritis，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）和磷脂内的（多）不饱和脂肪酰基残基的影响可以得到明确证实。然而，对于例如通过氧化修饰的脂质（具有反应性醛基）与丰富的蛋白质或糖胺聚糖的氨基反应而形成的产物，存在相当大的知识缺乏。此外，越来越多的人认识到，由ROS（损伤相关分子模式; DAMP）修饰的内源性分子激活细胞受体，伴随着炎症增加。我们假设这些已知但“未研究”的物质与炎性疾病的发病机制特别相关，即氧化化合物与炎性风湿性疾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。通过高分辨率色谱法、质谱法和NMR光谱法对这些产品进行详细表征是一项非常具有挑战性的任务，因为必须首先使方法适应这些分子。更具挑战性的是分析氧化脂肪酸/脂质与糖胺聚糖或模拟风湿性疾病中涉及的蛋白质（润滑素、聚集蛋白聚糖）活性位点的肽之间形成的反应产物。第二，将研究在第一个目标中鉴定的氧化分子类别在来自患有风湿性疾病的患者的滑液和来自滑液膜的提取物中与疾病的严重程度的关系。第三，我们将判断鉴定的氧化PL、GAG和蛋白质作为氧化DAMP对细胞免疫应答的相关性。对于这项任务，它们对T细胞，抗原呈递细胞和单核细胞从类风湿性滑膜的影响将在体外研究通过测量典型的炎症标志物，如干扰素-γ和选定的细胞因子释放的细胞。

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.