Synonyms

Pentraxin 3; TNF-inducible gene 14 protein; TSG-14

Historical Background

Innate immunity sets in evolution as the earliest line of defense against pathogens. Endowed with pivotal roles in activation and orientation of the adaptive immune response, this ancient system is deeply involved in a number of physiological and pathological processes, ranging from tissue homeostasis to cancer growth and development. Innate immunity comprises a cellular and humoral arm, the latter encompassing soluble pattern recognition molecules (PRMs) that recognize pathogen- and danger-associated molecular patterns (PAMPs and DAMPs, respectively) and initiate the immune response in coordination with the cellular arm (Bottazzi et al. 2010). Pentraxins are a phylogenetically conserved superfamily of soluble PRMs that, based on primary structure, is divided into two subfamilies: short and long pentraxins. Prototypes of the short pentraxins, C-reactive protein (CRP), and serum amyloid P (SAP) component were both identified at the beginning of last century as IL-6-induced, liver-derived acute phase proteins in human and mouse, respectively. First identified in the early 1990s as a TNF-α and IL-1β inducible gene in human endothelial cells and fibroblasts, PTX3 is a typical long pentraxin that features a C-terminal pentraxin-like domain (i.e., homologous to CRP and SAP) and a structurally unrelated N-terminal region, and differs from the short pentraxins in gene organization, chromosomal localization, cellular source, inducing stimuli and ligands. Locally made at sites of infection and inflammation by both somatic and immune cells, PTX3 cooperates with other soluble (e.g., complement) and cell-associated PRMs (e.g., scavenger and Toll-like receptors, TLRs) to provide nonredundant protective roles in the host resistance to selected pathogens, thus acting as a functional ancestor of antibodies (Bottazzi et al. 2010). Gene-targeted mice and genetic/epigenetic studies in humans indicate that PTX3 plays additional roles in inflammation and tissue remodeling (Doni et al. 2015). Recent studies pointed to a novel role of this long pentraxin as an extrinsic oncosuppressor gene that restrains the complement elicited tumor-promoting inflammation (Bonavita et al. 2015). The pleiotropic functions of PTX3 highlight the complexity of PRMs’ biology and set this long pentraxin as a paradigm of humoral innate immunity.

Gene Expression and Protein Structure

The ptx3 gene is remarkably conserved across evolutionary distant species in terms of sequence, structural organization and regulation, thus allowing evaluation of its pathophysiological roles in genetically modified animals. PTX3 expression is rapidly induced by several stimuli, such as inflammatory cytokines (e.g., IL-1β, TNF-α), TLR agonists, microbial moieties (e.g., LPS, OmpA, lipoarabinomannans), or intact microorganisms in several cell types, including myeloid dendritic cells (DCs), macrophages, endothelial cells, fibroblasts, kidney epithelial cells, synovial cells, chondrocytes, adipocytes, alveolar epithelial cells, glial cells, mesangial cells, and granulosa cells, but not in B and T lymphocytes neither in NK cells (Bottazzi et al. 2010). Interestingly, polymorphonuclear neutrophils (PMNs) lack de novo synthesis of PTX3; however, they store a constitutive stock of “ready-to-use” protein in specific granules, which is promptly released in response to TLR engagement and localizes in extracellular traps, DNA-rich structures that provide an effective antimicrobial microenvironment (Jaillon et al. 2007).

The human PTX3 protomer is a glycoprotein that comprises 381 amino acids, including a 17 amino acid signal peptide, an N-terminal region (18–178), and a C-terminal domain (179–381). A single N-glycosylation site occurs at Asn220 in the C-terminal pentraxin domain that is occupied by complex type oligosaccharides, mainly fucosylated and α-(2,3)-sialylated biantennary sugars. The glycosylation status of the protein, mainly through the sialic acid moiety, has been involved in a number of PTX3 functions in native immunity and inflammation (see below) (Inforzato et al. 2013). In addition to the multidomain organization, PTX3 has a complex quaternary structure with eight disulfide-linked protomers folding into an elongated and asymmetric molecule that contains a large and a small domain linked by a stalk region (Inforzato et al. 2010). The structural complexity and modular nature of the PTX3 protein likely provide a molecular framework to the rather broad spectrum of cellular and molecular targets of this long pentraxin and the diversity of its biological roles (Fig. 1).

PTX3, Fig. 1
figure 17179figure 17179

PTX3 and its functional roles in innate immunity and inflammation. PTX3 is produced by a number of both somatic and immune cells, including polymorphonuclear neutrophils (PMNs), macrophages (MФ), dendritic cells (DCs), endothelial cells, and fibroblasts at sites of inflammation and infection. The locally released protein is composed of eight identical subunits held together by disulfide bonds, each comprising distinct N- and C-terminal domains (in yellow and red, respectively) that fold into an asymmetric molecule with two differently sized domains linked by a short stalk. The structural complexity of the PTX3 protein provides a framework for recognition of a multiplicity of ligands, including microorganisms, complement components, and modified self-antigens (e.g., apoptotic cells). When bound to microbes and apoptotic cells, PTX3 promotes their disposal by phagocytes, via a tight molecular crosstalk with the complement system, and modulates the complement-dependent inflammation. The interaction of PTX3 with viruses can either facilitate viral entry (e.g., arthritogenic alphaviruses) or lead to viral neutralization and inactivation (e.g., influenza virus A, HAV, and cytomegalovirus, CMV). PTX3 exerts additional anti-inflammatory roles in a complement-independent fashion, for example, by reducing leukocyte recruitment at inflamed sites through a specific interaction with P-selectin. As a component of the extracellular matrix (ECM), PTX3 participates in the remodeling of hyaluronan (HA) and fibrin networks at sites of inflammation and tissue injury. Moreover, PTX3 acts as an extrinsic oncosuppressor gene, where it can regulate complement-dependent tumor-promoting inflammation reducing tumor growth. However, pro-tumorigenic effects have been reported for this long pentraxin, thus questioning the real role of PTX3 in cancerogenesis. (UPEC, uropathogenic strains of E. coli; MBL, mannose-binding lectin; C4bp, C4b-binding protein)

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Crosstalk with the Complement System

PTX3 can modulate all three pathways of complement (i.e., classic, CP, lectin, LP, and alternative, AP), a property that is shared with the short pentraxins. C1q (i.e., the recognition unit of the CP) is the first identified ligand of PTX3, where this interaction may have opposite functional outcomes depending on whether PTX3 is bound to a surface or free in solution (with enhanced or inhibited C3 and C4 deposition, respectively). Indeed, the interaction of PTX3 with C1q in the fluid phase prevents C1q binding and C3 deposition onto apoptotic cells (i.e., a cellular ligand of PTX3) as well as the C1q-mediated phagocytosis of these cells by DCs. In contrast, when preincubated with apoptotic cells, PTX3 enhances deposition of both C1q and C3 on the cell surface. On this line, membrane-associated PTX3 acts as an “eat-me” molecule in promoting phagocytosis of late apoptotic neutrophils, as opposed to the soluble form of PTX3 that inhibits this process. This view is supported by data from an in vivo murine model of systemic lupus erythematosus, where PTX3 fosters the rapid clearance of apoptotic T cells by peritoneal macrophages. PTX3 interacts with members of the LP, namely, ficolin-1, ficolin-2, and mannose-binding lectin (MBL), where, for example, the engagement of ficolin-2 and MBL leads to a functional cooperation that promotes recruitment of either molecule onto selected microbes (i.e., Aspergillus fumigatus and Candida albicans) and synergistically amplifies the LP response. Also, PTX3 recognizes the major soluble inhibitors of the CP/LP and AP, C4bp and factor H, respectively. When bound to apoptotic cells, this long pentraxin recruits factor H and, in this way, limits complement activation, increases phagocytosis of apoptotic cells, and reduces the inflammatory response (i.e., by inhibiting the downstream pathway of complement activation that generates the anaphylatoxins C3a and C5a). Furthermore, recognition of factor H by PTX3 is impaired by factor H mutations and autoantibodies associated with atypical hemolytic uremic syndrome (aHUS), which might result into exacerbation of local complement-mediated inflammation, a distinctive trait of this disease. Similar to factor H, PTX3 recruits C4bp onto apoptotic cells and extracellular matrix (ECM), increasing the rate of C4b inactivation and reducing deposition of the complement terminal complex. Based on these evidences, it appears that PTX3 might contribute to the noninflammatory, phagocytic removal of apoptotic cells, in this way restraining the release of autoantigens and the onset of autoimmune responses (Doni et al. 2012). Protein glycosylation plays a “tuning” role in the interaction of PTX3 with its complement ligands and the corresponding functional outcomes. For example, desialylation increases the binding of PTX3 to C1q, while abrogating its recognition by ficolin-1; also, deglycosylation strongly impairs the association of this long pentraxin with factor H (Inforzato et al. 2013).

Roles in Antimicrobial Immunity

Since its discovery, PTX3 has been increasingly recognized as a nonredundant protective factor in a number of fungal and bacterial infections. For example, PTX3-deficient mice are susceptible to invasive aspergillosis, a life-threatening infection among immunocompromised individuals primarily caused by the opportunistic fungus Aspergillus fumigatus (AF). This susceptibility is associated with a low protective T helper 1 (Th1) antifungal response coupled with an inappropriate Th2 response. Administration of the exogenous protein rescues the Th1 phenotype and has therapeutic efficacy in several models of IA in immunocompromised hosts (Garlanda et al. 2002). Current literature indicates that PTX3 has opsonic activity towards AF, and enhances recognition, phagocytosis, and killing of fungal conidia by immune cells, mainly PMNs, via complement and Fc receptors (FcRs) pathways (Moalli et al. 2010). PTX3 has therapeutic activity in animal models of chronic lung infection by Pseudomonas aeruginosa, a major cause of morbidity and mortality in cystic fibrosis patients. Also in this condition, the PTX3-dependent recognition and phagocytosis of the pathogen involves a tight interplay with complement and FcRs. Similar protective roles have been reported for this long pentraxin in the innate resistance to uropathogenic strains of Escherichia coli (UPEC), where again it acts as an opsonin by facilitating recognition and phagocytosis of bacteria by immune cells. Furthermore, PTX3 opsonizes Neisseria meningitides and decreases the bacterial load in an infant rat model of meningococcal meningitis. Interestingly, in this model, PTX3 amplifies the antibody response to vaccination, thus acting as a “natural adjuvant” of adaptive immunity. PTX3 binds to the outer membrane protein A (KpOmpA) of Klebsiella pneumoniae and amplifies the complement-dependent response to this bacterium via functional cooperation with cell-associated PRMs, including the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the scavenger receptor expressed by endothelial cell-I (SREC-I), and TLR2 (Jeannin et al. 2005).

Evidence is growing to suggest that PTX3 can mediate antiviral activities both in vitro and in vivo against human and murine cytomegalovirus (HCMV and MCMV, respectively) and influenza virus type A (H3N2 strain). PTX3 binds both HCMV and MCMV and reduces viral entry and infectivity in DCs in vitro. Consistently, PTX3-deficient mice are more susceptible to MCMV infection than their wild-type littermates, and PTX3 protects susceptible animals from MCMV primary infection and reactivation in vivo as well as Aspergillus superinfection (Bozza et al. 2006). Also, human and murine PTX3 bind influenza virus H3N2, where this interaction involves the viral hemagglutinin glycoprotein and the sialic acid residues present on the glycosidic moiety of the protein. PTX3 inhibits the virus-induced hemagglutination and viral neuraminidase activity and neutralizes the virus infectivity (Reading et al. 2008). As opposed to these antiviral activities, recent studies indicate that PTX3 can promote rather than inhibit virus entry and replication in target cells. This is the case of arthritogenic alphaviruses (i.e., chikungunya and Ross River viruses), which highlights a potential contribution of this long pentraxin to the pathogenesis of alphaviral diseases (Foo et al. 2015).

Elevated circulating levels of PTX3 have been found in several systemic infections. For example, in a cohort of adult patients admitted to Intensive Care Unit with positive blood culture for the most common causative microorganisms in community-acquired bacteremia (Staphylococcus aureus, Streptococcus pneumoniae, β-hemolytic streptococci, or E. coli), nonsurvivors showed the highest PTX3 titers on days 1–4, while CRP levels were not predictive of mortality. Evaluation of the PTX3 concentration during a systemic inflammatory response may therefore improve patient risk assessment and management. Additional studies pointed to PTX3 as a prognostic/diagnostic marker in a number of infectious diseases, including Dengue virus infections, pulmonary tuberculosis (TB), leptospirosis, meningococcal diseases, pulmonary aspergillosis, and urinary tract infections caused by UPEC. Most interestingly, PTX3 genetic variants have been found to be associated with several infections in humans, including pulmonary tuberculosis, cystic fibrosis-related Pseudomonas aeruginosa infections, UPEC burdens, and IA in hematopoietic stem cells transplanted patients. Overall, these reports support prophylactic, therapeutic, and diagnostic applications of PTX3 in infectious diseases (Garlanda et al. 2016).

Roles in Inflammation and Tissue Remodeling

Given the production of PTX3 by myeloid cells in response to primary inflammatory cytokines, it is expected that this long pentraxin, similarly to the short ones, plays a role in the regulation of inflammation. In this regard, PTX3 can affect cell recruitment at inflamed sites through the interaction with the adhesion molecule P-selectin, which is mediated by the sialylated oligosaccharides of the protein. Upon binding to P-selectin, PTX3 limits early neutrophil recruitment in response to inflammatory stimuli. Indeed, PTX3-deficient mice show enhanced neutrophil infiltration in models of pleurisy, acute lung injury, and ischemia/reperfusion-induced kidney damage, strongly suggesting that this long pentraxin takes part in a negative feedback loop that regulates PMN extravasation (Deban et al. 2010).

Recent observations indicate that PTX3 is an important player in wound healing. In different animal models of tissue damage (i.e., skin wound healing, sterile liver and lung injury, arterial thrombosis) genetic deficiency of PTX3 causes increased fibrin deposition and persistence and larger clot formation, likely due to impaired pericellular fibrinolysis and directional migration of macrophages (i.e., a source of PTX3 in vitro and in vivo). It has been proposed that the macrophage-released PTX3 participates in fibrinolysis by promoting the plasminogen-dependent resorption of the provisional fibrin matrix. Interestingly, this activity is maximal at acidic pH, a condition that normally occurs at sites of tissue injury and repair, thus indicating that pH can set PTX3 in a tissue repair mode (Doni et al. 2015).

A role for PTX3 is described in different conditions of ischemia and reperfusion, including acute myocardial infarction (AMI). In humans and mice, PTX3 is rapidly produced during AMI and ptx3−/− mice have higher no-reflow area, increased neutrophil infiltration, decreased number of capillaries, and increased number of apoptotic cardiomyocytes (Salio et al. 2008). In addition, higher C3 deposition was observed in the lesioned tissues of these animals. Furthermore, larger aortic lesions and a more pronounced inflammatory profile have been reported in a model of atherosclerosis in apolipoprotein E- and PTX3-deficient mice. Also, in a murine model of coxsackievirus B3-dependent myocarditis, PTX3-deficiency was associated with increased heart injury and cardiomyocyte apoptosis. However, in a mouse model of intestinal ischemia and reperfusion, genetic depletion of PTX3 was associated to lower inflammation and reduced lethality. Moreover, in a model of ventilator-induced lung injury, PTX3 overexpression resulted in increased inflammatory response, and the protein was found to inhibit acetylcholine-induced vasorelaxation. Thus, in the context of reperfusion and injury, PTX3 could exert dual opposite roles, being protective or deleterious depending on tissue district (Garlanda et al. 2016).

Besides its presence in the blood as a circulating protein, PTX3 can be detected in the ECM. Notably, PTX3 is an essential component of the viscoelastic hyaluronan (HA)-rich matrix that forms around the oocyte in the preovulatory follicle and is necessary for fertilization in vivo. Therein, it provides matrix stability by establishing multivalent contacts to the HA-binding proteins TSG-6 (TNF-stimulated gene 6 protein) and the serum proteoglycan IαI (inter-alpha-trypsin inhibitor). Indeed, genetic or functional deletion of PTX3, TSG-6, or IαI leads to impaired cumulus matrix formation and severe subfertility. Interestingly, PTX3, HA, TSG-6, and IαI are all present in the endothelial ECM, where they regulate different aspects of the endothelium biology, including pathological conditions like atherosclerosis (Inforzato et al. 2011).

In line with its roles in inflammation, PTX3 acts as an acute phase protein and its plasmatic levels increase rapidly (with a maximum at 6–8 h) from a basal value of approximately 2 ng/ml in healthy subjects, to hundreds of nanograms in inflammatory conditions. The production of PTX3 by different cell types guarantees a local effect of the molecule, a major difference to CRP that is made by the liver and acts systemically. In this regard, based on the aforementioned observation that PTX3 plasma levels increase in AMI patients, this long pentraxin has been identified as the only independent predictor of mortality within 3 months of the acute event. Furthermore, high systemic levels of PTX3 are associated with increased risk of cardiovascular morbidity and mortality, which provides further insights into the role of inflammation in these pathological conditions. Additional studies have reported higher titers of PTX3 in the plasma of chronic kidney disease and hemodialysis patients, where this protein appears to be a promising biomarker (Garlanda et al. 2016).

PTX3 in Cancer

It is generally accepted that inflammation plays an essential role in tumor development; thus, it is expected that PTX3, as a regulator of inflammation, could play a role in cancer. Indeed, PTX3 deficiency causes increased susceptibility to mesenchymal and epithelial carcinogenesis in models of 3-methylcholanthrene (3-MCA)-induced carcinogenesis and 7,12-dimethylbenz [α] anthracene/terephtalic acid (DMBA/TPA)-induced skin carcinogenesis. Enhanced levels of tumor-associated macrophages, higher production of pro-inflammatory cytokines, increased DNA damage, higher angiogenesis and C3 deposition were observed in these models. PTX3 regulation of C3 deposition on tumor cells occurs through recruitment of factor H, suggesting that the unleashed complement activation observed in ptx3−/− mice plays a crucial role in promotion of an inflammatory, pro-tumoral microenvironment (Bonavita et al. 2015).

It is long known that PTX3 acts as a natural antagonist of the fibroblast growth factors 2 and 8b (FGF2 and FGF8b, respectively), which play crucial roles in cancer. In a recent report, human PTX3 overexpression in transgenic mice was found to inhibit tumor growth, angiogenesis, and metastasis in FGF-dependent tumor models. Most interestingly, a small-molecule derived from the FGF2-binding sequence of PTX3 was described to act as an extracellular trap for this factor, with important implications in cancer therapy (Ronca et al. 2015).

In humans, PTX3 expression is increased in different cancers, including glioma, lung cancer, liposarcoma, prostate and pancreatic carcinoma, breast cancer (Infante et al. 2016). Polymorphisms of the ptx3 gene are associated with circulating levels of the protein and risk to develop hepatocellular carcinoma in subjects infected with hepatitis C virus. In a cohort of ovarian cancer patients, the ptx3 gene emerged in a poor prognosis stromal genetic signature. Furthermore, epigenetic regulation of PTX3 expression has been investigated in several human cancers, including esophageal squamous cell carcinoma, colorectal cancer, leyomiosarcomas, and desmoid tumors, where promoter and other regulatory regions of the ptx3 gene were found to be highly methylated (i.e., with reduced gene expression) (Bonavita et al. 2015). Collectively, these data suggest that PTX3 acts as an extrinsic oncosuppressor gene and exemplify the connection between inflammation and cancer perceived in the last years (Mantovani et al. 2008).

Beside the role as oncosuppressor, few reports outlined a pro-tumoral role of PTX3. In gastric cancer and head and neck tumors, PTX3 promotes tumor cell migration and invasion, while in human glioma the protein sustains tumor cell proliferation. In addition, in gastric cancer PTX3 silencing suppresses cancer-related inflammation. These contrasting results suggest that further studies are necessary to define the real significance of PTX3 in the regulation of tumor-associated inflammation (Garlanda et al. 2016).

Summary

The humoral arm of innate immunity is a complex ensemble of structurally and functionally diverse molecules belonging to different families (ficolins, collectins, complement). Pentraxins and PTX3 in particular are paradigmatic soluble PRMs. As such, they interact and synergize in microbial recognition and disposal and, in general terms, can be viewed as evolutionarily ancient, antibody-like molecules. Like antibodies, PTX3 and other fluid phase PRMs have regulatory functions in inflammation and tissue remodeling. The recent discovery that PTX3 acts as an extrinsic oncosuppressor in selected tumors opens new perspectives on the role of the innate immune system in tumor progression. In this regard, it is expected that the emerging vistas on the humoral arm of this system (with PTX3 as a paradigm) will pave the way to unforeseen diagnostic and therapeutic translational tools.