The proteomic profile of circulating pentraxin 3 (PTX3) complex in sepsis demonstrates the interaction with azurocidin 1 and other components of neutrophil extracellular traps

Pentraxin 3 (PTX3)1 is a secretory protein classified as a long pentraxin subfamily member of the pentraxin family. The pentraxin family proteins, which are evolutionarily conserved multimeric pattern recognition receptors and share a pentraxin-like domain in the C terminus, are recognized as key components of humoral innate immunity (1). PTX3 has a unique 200-amino acid domain in its N terminus and is known to play multiple roles, including the regulation of inflammatory reactions, innate resistance to pathogens, and female fertility (2). PTX3 is expressed in a variety of cells at inflammatory sites (3) and is also stored in neutrophil-specific granules (4). The stored PTX3 in neutrophils is released into the extracellular space and localizes to neutrophil extracellular traps (NETs) (4), which are extracellular fibers consisting of DNA, histones, and antimicrobial proteins that capture and kill pathogens (5). PTX3 is useful as a diagnostic marker of vascular damage and infections (6). In septic patients, the circulating PTX3 concentration increases to an especially high level (7). Sepsis is one of the major causes of death in developed countries (8). Despite extensive studies, an effective treatment is not yet available. During the past few decades, sepsis has come to be recognized as a heterogeneous, complex, and dynamic syndrome caused by imbalances in the inflammatory network (9). It has been accepted that sepsis develops through two stages: an initial pro-inflammatory response, defined as the systemic inflammatory response syndrome, and a concomitant anti-inflammatory phase, referred to as the compensatory anti-inflammatory response syndrome. The pro-inflammatory response is initiated by the PRRs in immune cells recognizing molecules originating from infectious pathogens termed pathogen-associated molecular patterns and from inflammatory cells or tissues known as damage-associated molecular patterns (10). PTX3, a soluble PRR, has been shown to bind certain pathogens, complement components, and even to other PRRs in a calcium ion-dependent or -independent manner (1, 2). In addition to its pro-inflammatory activity, PTX3 also has been shown to play a role in protecting against severe inflammatory reactions, such as animal sepsis models (11), seizure-induced neurodegeneration (12), and acute myocardial infarction (13). As a marker of sepsis, plasma PTX3 exhibits a good correlation with mortality (7). An in vivo study showed that PTX3 transgenic mice are resistant to endotoxic shock and polymicrobial sepsis (11). Although negative feedback mediation of inflammation has been postulated (14), the actual roles of PTX3 in sepsis are not fully understood. One of the approaches to the understanding of the mechanisms is the proteomic identification of the specific PTX3 ligands. The MS-based clinical proteomics approach is widely used both as a biomarker discovery and for verification purposes (15). However, it is generally considered that extensive fractionation is required to identify a new biomarker in biofluids (16) because of the wide dynamic range of proteins in blood and biofluids (17). On the other hand, affinity purification together with the MS strategy is a feasible approach to the identification of protein interactions, which combines tagged protein purification and quantitative proteomics using protein stable isotope labeling (18, 19). Here again, however, there are also technical difficulties in practically adapting this strategy to the clinical setting such as sepsis. One of the solutions to these technical barriers is the usage of antibodies. Immunopurification enables isolation of the protein of interest in a simple and effective way. In fact, antibody-based proteomics has been used in the discovery and confirmation of biomarkers in cancer (20). Thus, to better understand the molecular mechanisms of PTX3, we investigated PTX3 ligands by immunopurification of native PTX3 from septic patient fluids and shotgun proteomics for label-free relative quantitation. From the proteomic analysis, we found novel interactants, including some of the components of NETs, as well as known PTX3 ligands such as complement and extracellular matrix proteins. Further investigation revealed that azurocidin 1 (21), a bactericidal protein that localizes to NETs, was one of these direct PTX3 interacting partners that act through the oligomer N-terminal domain of PTX3 in a calcium ion-dependent manner. The interaction between PTX3 and components of NETs might play a protective role in sepsis. This strategy of targeted proteomics provides a useful method for detecting low levels of diagnostic biomarkers.