The Proteomic Profile of Circulating Pentraxin 3 (PTX3) Complex in Sepsis Demonstrates the Interaction with Azurocidin 1 and Other Components of Neutrophil Extracellular Traps

Abstract

Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (∼200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (KD = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target. Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (∼200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (KD = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target. Pentraxin 3 (PTX3) 1The abbreviations used are:PTX3pentraxin 3AZU1azurocidin 1GOGene OntologyNETneutrophil extracellular trapMPOmyeloperoxidasePMAphorbol myristate acetatePRRpattern recognition receptor. 1The abbreviations used are:PTX3pentraxin 3AZU1azurocidin 1GOGene OntologyNETneutrophil extracellular trapMPOmyeloperoxidasePMAphorbol myristate acetatePRRpattern recognition receptor. 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 (1Bottazzi B. Doni A. Garlanda C. Mantovani A. An integrated view of humoral innate immunity: Pentraxins as a paradigm.Annu. Rev. Immunol. 2010; 28: 157-183Crossref PubMed Scopus (438) Google Scholar). 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 (2Mantovani A. Garlanda C. Doni A. Bottazzi B. Pentraxins in innate immunity: From C-reactive protein to the long pentraxin PTX3.J. Clin. Immunol. 2008; 28: 1-13Crossref PubMed Scopus (336) Google Scholar). PTX3 is expressed in a variety of cells at inflammatory sites (3Garlanda C. Bottazzi B. Bastone A. Mantovani A. Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility.Annu. Rev. Immunol. 2005; 23: 337-366Crossref PubMed Scopus (715) Google Scholar) and is also stored in neutrophil-specific granules (4Jaillon S. Peri G. Delneste Y. Frémaux I. Doni A. Moalli F. Garlanda C. Romani L. Gascan H. Bellocchio S. Bozza S. Cassatella M.A. Jeannin P. Mantovani A. The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps.J. Exp. Med. 2007; 204: 793-804Crossref PubMed Scopus (421) Google Scholar). The stored PTX3 in neutrophils is released into the extracellular space and localizes to neutrophil extracellular traps (NETs) (4Jaillon S. Peri G. Delneste Y. Frémaux I. Doni A. Moalli F. Garlanda C. Romani L. Gascan H. Bellocchio S. Bozza S. Cassatella M.A. Jeannin P. Mantovani A. The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps.J. Exp. Med. 2007; 204: 793-804Crossref PubMed Scopus (421) Google Scholar), which are extracellular fibers consisting of DNA, histones, and antimicrobial proteins that capture and kill pathogens (5Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6121) Google Scholar). PTX3 is useful as a diagnostic marker of vascular damage and infections (6Presta M. Camozzi M. Salvatori G. Rusnati M. Role of the soluble pattern recognition receptor PTX3 in vascular biology.J. Cell Mol. Med. 2007; 11: 723-738Crossref PubMed Scopus (145) Google Scholar). In septic patients, the circulating PTX3 concentration increases to an especially high level (7Mauri T. Bellani G. Patroniti N. Coppadoro A. Peri G. Cuccovillo I. Cugno M. Iapichino G. Gattinoni L. Pesenti A. Mantovani A. Persisting high levels of plasma pentraxin 3 over the first days after severe sepsis and septic shock onset are associated with mortality.Intensive Care Med. 2010; 36: 621-629Crossref PubMed Scopus (124) Google Scholar). pentraxin 3 azurocidin 1 Gene Ontology neutrophil extracellular trap myeloperoxidase phorbol myristate acetate pattern recognition receptor. pentraxin 3 azurocidin 1 Gene Ontology neutrophil extracellular trap myeloperoxidase phorbol myristate acetate pattern recognition receptor. Sepsis is one of the major causes of death in developed countries (8Martin G.S. Mannino D.M. Eaton S. Moss M. The epidemiology of sepsis in the United States from 1979 through 2000.N. Engl. J. Med. 2003; 348: 1546-1554Crossref PubMed Scopus (4818) Google Scholar). 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 (9Rittirsch D. Flierl M.A. Ward P.A. Harmful molecular mechanisms in sepsis.Nat. Rev. Immunol. 2008; 8: 776-787Crossref PubMed Scopus (883) Google Scholar). 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 (10Xiang M. Fan J. Pattern recognition receptor-dependent mechanisms of acute lung injury.Mol. Med. 2010; 16: 69-82Crossref PubMed Scopus (77) Google Scholar). 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 (1Bottazzi B. Doni A. Garlanda C. Mantovani A. An integrated view of humoral innate immunity: Pentraxins as a paradigm.Annu. Rev. Immunol. 2010; 28: 157-183Crossref PubMed Scopus (438) Google Scholar, 2Mantovani A. Garlanda C. Doni A. Bottazzi B. Pentraxins in innate immunity: From C-reactive protein to the long pentraxin PTX3.J. Clin. Immunol. 2008; 28: 1-13Crossref PubMed Scopus (336) Google Scholar). 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 (11Souza D.G. Soares A.C. Pinho V. Torloni H. Reis L.F. Teixeira M.M. Dias A.A. Martins M.T. Increased mortality and inflammation in tumor necrosis factor-stimulated gene-14 transgenic mice after ischemia and reperfusion injury.Am. J. Pathol. PubMed Scopus Google Scholar), T. D. Bottazzi B. Peri G. Garlanda C. Mantovani A. A. of the long pentraxin PTX3 in the after for a role in PubMed Scopus Google Scholar), and acute M. S. N. F. V. M. F. Garlanda C. Mantovani A. of the long pentraxin PTX3 in acute 2008; PubMed Scopus Google Scholar). a marker of sepsis, plasma PTX3 a with mortality (7Mauri T. Bellani G. Patroniti N. Coppadoro A. Peri G. Cuccovillo I. Cugno M. Iapichino G. Gattinoni L. Pesenti A. Mantovani A. Persisting high levels of plasma pentraxin 3 over the first days after severe sepsis and septic shock onset are associated with mortality.Intensive Care Med. 2010; 36: 621-629Crossref PubMed Scopus (124) Google Scholar). An in study showed that PTX3 transgenic mice are to shock and sepsis (11Souza D.G. Soares A.C. Pinho V. Torloni H. Reis L.F. Teixeira M.M. Dias A.A. Martins M.T. Increased mortality and inflammation in tumor necrosis factor-stimulated gene-14 transgenic mice after ischemia and reperfusion injury.Am. J. Pathol. PubMed Scopus Google Scholar). of inflammation has been L. M. Moalli F. M. V. Cuccovillo I. Bastone A. M. S. Doni A. Garlanda C. S. Salvatori G. M. V. B. G. C. Bottazzi B. Mantovani A. of by the long pentraxin Immunol. 2010; 11: PubMed Scopus Google Scholar), the roles of PTX3 in sepsis are not understood. of the to the of the mechanisms is the proteomic of the specific PTX3 The proteomics is used as a and for and 2010; PubMed Scopus Google Scholar). is that extensive is to a in P. B. 2010; 28: PubMed Scopus Google Scholar) of the dynamic of proteins in and The plasma and diagnostic PubMed Scopus Google Scholar). the other affinity with the is a to the of protein which protein and proteomics using protein A.C. M. B. of protein complexes using Rev. Mol. Cell 2007; 8: PubMed Scopus Google Scholar, L. of protein interaction of protein complexes using affinity and 2010; PubMed Scopus Google Scholar). however, are also in to the such as of the to is the of of the protein of in a and effective In proteomics has been used in the and of in F. molecular in Rev. 2010; PubMed Scopus Google Scholar). to the molecular mechanisms of PTX3, we PTX3 ligands by immunopurification of PTX3 from septic patient and shotgun proteomics for label-free relative From the proteomic we including some of the components of as well as known PTX3 ligands such as complement and extracellular matrix revealed that azurocidin 1 as a inflammatory 2003; PubMed Scopus Google Scholar), a bactericidal protein that localizes to one of direct PTX3 that through the oligomer N-terminal domain of PTX3 in a calcium ion-dependent The interaction between PTX3 and components of play a role in of targeted proteomics a useful for levels of diagnostic azurocidin 1 and myeloperoxidase from complement from monoclonal antibody against PTX3 that generated as A. 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T. of and PubMed Scopus Google Scholar). that of a an and with an used for of the into a to an for and the the trap with the a by the The used and The developed at the of with a concentration of from to for from to for 1 at for from to for 1 and with for into the via the that the with an The and the of the to or The in a in which the with a of to the of The by an with at a of = for the by trap in the using the with of and 104 for and also a dynamic that the of the of in the of with an of and of and The with the the of with against the as the and for of to two The with a of 3 and of into the by of by the of of of the N terminus, of and of used for and relative accepted be at as by the A. to the of by and PubMed Scopus Google Scholar). accepted be at and at two identified by the A. for proteins by 2003; PubMed Scopus Google Scholar). a a L. N. V. label-free of protein in from and 8: PubMed Scopus Google Scholar). that and not be to the of relative protein performed using spectral the of identified in of proteins that performed an identified using the from the of the spectral of immunopurification to the spectral of immunopurification in the candidate proteins its unique and the in at one The Gene Ontology with Gene defined the as proteins in and 1 with of of protein in at The with of in for at the of PTX3 with in with to well and for 1 at a of antibody or antibody with in and for 1 at The developed with of for at and with of at the with the of or to of the performed using by of the performed using a of PTX3 the performed with an using a The at at using with and as the of the by a of for of PTX3 with for of by a of in the used as a of PTX3 by the of azurocidin 1 at for of association by of of the by an of of 1 by of the of the with or 3 the using PTX3 expressed and as A. M. S. T. G. I. H. M. T. T. T. 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T. of and PubMed Scopus Google Scholar). with protein magnetic of the to The in the and using the immunopurification with the monoclonal of the by with the antibody for of the and and The of PTX3 from of in the of From the of the protein and of the the as high to shotgun proteomics of PTX3 from patient from the of the plasma and with of PTX3 from the of the plasma and with of PTX3. in a proteomics performed using PTX3 complex From the 104 proteins as PTX3-interacting protein using label-free relative quantitation via spectral L. of protein interaction of protein complexes using affinity and 2010; PubMed Scopus Google Scholar, L. N. V. label-free of protein in from and 8: PubMed Scopus Google Scholar) and proteins, the known PTX3 ligands and PTX3 proteins, such as complement components and extracellular matrix proteins, and Notably, certain proteins that are components of neutrophil granules G. Ward of neutrophil 2005; PubMed Scopus Google Scholar) and D. M. U. Goosmann C. V. 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Doni A. Garlanda C. Mantovani A. An integrated view of humoral innate immunity: Pentraxins as a paradigm.Annu. Rev. Immunol. 2010; 28: 157-183Crossref PubMed Scopus (438) Google Scholar). PTX3 ligands full-length PTX3 for PTX3 binding (1Bottazzi B. Doni A. Garlanda C. Mantovani A. An integrated view of humoral innate immunity: Pentraxins as a paradigm.Annu. Rev. Immunol. 2010; 28: 157-183Crossref PubMed Scopus (438) Google Scholar). we N-terminal and PTX3 proteins to the PTX3 domain for binding to The multimeric of protein with with a A. C. M. V. A. Bottazzi B. Mantovani A. Salvatori G. The long pentraxin PTX3 an with two binding sites for 2010; PubMed Scopus Google Scholar). The binding showed that AZU1 bound to the N-terminal domain of PTX3 exhibited binding in the N-terminal and of PTX3, the domain exhibited a the oligomer of the N-terminal domain of PTX3 AZU1 binding using protein which is a protein that through the N-terminal domain of PTX3 the binding to the PTX3 N-terminal domain N-terminal domain PTX3 its A. C. M. V. A. Bottazzi B. Mantovani A. Salvatori G. The long pentraxin PTX3 an with two binding sites for 2010; PubMed Scopus Google Scholar). The oligomer in protein with also with a A. C. M. V. A. Bottazzi B. Mantovani A. Salvatori G. The long pentraxin PTX3 an with two binding sites for 2010; PubMed Scopus Google Scholar). The showed that the N-terminal domain of a PTX3 in which the at and to the binding to AZU1 of N-terminal domain PTX3 oligomer for binding to azurocidin of the PTX3 N-terminal domain and the N-terminal domain with of the with expressed by of proteins with and binding of the N-terminal domain of PTX3 to AZU1 to the PTX3 The antibody antibody of the The are from of to the binding of in the we performed an we generated a monoclonal antibody against AZU1 and the by The monoclonal antibody specific of full-length AZU1 in cells cells into a neutrophil and neutrophils also an AZU1 in the neutrophil by co-localization of AZU1 and PTX3 the from the neutrophils with of the in diagnostic or has been to plasma proteins the of or even Despite the of is to the components of plasma been to an proteomics to complexes of proteins T. Y. S. T. H. M. T. S. T. of and PubMed Scopus Google Scholar). to an of the PTX3 complex in septic plasma and using a specific monoclonal antibody PTX3, which is in plasma ng/ml) (6Presta M. Camozzi M. Salvatori G. Rusnati M. Role of the soluble pattern recognition receptor PTX3 in vascular biology.J. Cell Mol. Med. 2007; 11: 723-738Crossref PubMed Scopus (145) Google Scholar) and is known to be elevated to as high as in sepsis, with the is to the of the (7Mauri T. Bellani G. Patroniti N. Coppadoro A. Peri G. Cuccovillo I. Cugno M. Iapichino G. Gattinoni L. Pesenti A. Mantovani A. Persisting high levels of plasma pentraxin 3 over the first days after severe sepsis and septic shock onset are associated with mortality.Intensive Care Med. 2010; 36: 621-629Crossref PubMed Scopus (124) Google Scholar). of sepsis been the or for C. Sepsis 2010; PubMed Scopus Google Scholar). 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M. pentraxin 3 (PTX3) and by neutrophils in and in PubMed Scopus Google Scholar), exhibited binding to PTX3 The other candidate proteins we not binding to PTX3 be affinity interaction proteins or AZU1 and are direct PTX3-interacting protein in sepsis, we the binding mechanisms in AZU1 exhibited calcium ion-dependent which in a manner and also high affinity binding to PTX3. AZU1 binding the PTX3 N-terminal domain as also the for and PTX3 interaction A. C. M. V. A. Bottazzi B. Mantovani A. Salvatori G. The long pentraxin PTX3 an with two binding sites for 2010; PubMed Scopus Google Scholar) suggests a binding for the biochemical the direct binding of AZU1 to PTX3, we PTX3 and AZU1 associated in An monoclonal antibody that we shown to specifically the AZU1 and AZU1 in neutrophils as a inflammatory 2003; PubMed Scopus Google Scholar, J. a with specific Clin. 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