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

Subject Area Rheumatology
Analytical Chemistry
Term from 2014 to 2019
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 264172152
 
Final Report Year 2019

Final Report Abstract

Destruction of weight-bearing or small finger joints, which can be caused by 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 hypochlorus acid (HOCl) and hydroxyl radicals (HO•), is an important hallmark of inflammatory diseases, particularly of 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. Our proposal was based on the hypothesis that oxidized compounds are particularly relevant for the pathogenesis of inflammatory rheumatic diseases and that these compounds are, thus, of diagnostic and therapeutic importance. We have dealt with these aspects on different levels of complexity. First, we synthesized different oxidized and chlorinated fatty acids and phospholipids. It was extremely surprising for us that the reaction products between HOCl and phospholipids, on the one hand, and free fatty acids, on the other hand, were completely different: while phosphatidylcholines (as the most abundant lipids in body fluids) yielded the expected chlorohydrins, the reaction pattern of free fatty acids was much more complex: in addition to chlorohydrins, there were also dimeric and trimeric products. It is important to note that there were in addition to ester linkages also ether linkages. The detailed characterization of these products by chromatography, mass spectrometry and NMR spectroscopy was a quite challenging task. We could also show that oxidized lipids such as phosphatidylcholine chlorohydrins are less susceptible towards phospholipase A2 digestion in comparison to the non-modified lipids. Therefore, it is likely that these compounds accumulate in synovial fluids under inflammatory conditions and may be useful as disease markers. In parallel, we searched for disease-specific protein modifications initiated by oxidative or nitrosative stress in synovial fluid, serum, and plasma samples obtained from RA and non-RA patients. While oxidative and nitrosative modifications including reactive carbonyl groups, lipid peroxidation products, and nitro-tyrosine were not elevated in RA samples, the modification degrees of several protein glycation sites were significantly increased in sera obtained from RA patients. In total five modification sites were identified in three proteins, which allowed a good separation of diseased and non-diseased groups. The most promising candidate provided a very promising sensitivity of 98% and a high specificity of 90%. While these data have been validated for samples collected at the Leipzig University hospital, further studies using serum samples from other hospitals have to show if the identified modification indeed represent promising candidate biomarkers. When the effects of oxidative stress on cellular function were investigated in monocytes, we found that conditions favoring the generation of reactive oxygen species were always accompanied by an augmented response towards pro-inflammatory stimuli. Oxidized biomolecules, in turn, were found to stimulate an increased pro-inflammatory response in monocytes, which emphasizes the physiological relevance of oxDAMPs.

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