Role of the soluble pattern recognition receptor PTX3 in vascular biology

Pentraxins act as soluble pattern recognition receptors with a wide range of functions in various pathophysiological conditions. The long-pentraxin PTX3 shares the C-terminal pentraxin-domain with short-pentraxins C-reactive protein and serum amyloid P component and possesses an unique N-terminal domain. These structural features suggest that PTX3 may have both overlapping and distinct biological/ligand recognition properties when compared to short-pentraxins. PTX3 serves as a mechanism of amplification of inflammation and innate immunity. Indeed, vessel wall elements produce high amounts of PTX3 during inflammation and the levels of circulating PTX3 increase in several pathological conditions affecting the cardiovascular system. PTX3 exists as a free or extracellular matrix-associated molecule and it binds the complement fraction C1q. PTX3 binds also apoptotic cells and selected pathogens, playing a role in innate immunity processes. In endothelial cells and macrophages, PTX3 upregulates tissue factor expression, suggesting its action as a regulator of endothelium during thrombogenesis and ischaemic vascular disease. Finally, PTX3 binds the angiogenic fibroblast growth factor-2, thus inhibiting its biological activity. Taken together, these properties point to a role for PTX3 during vascular damage, angiogenesis, atherosclerosis, and restenosis.     

Pentraxins in innate immunity: lessons from PTX3

The innate immune system constitutes the first line of defence against microorganisms and plays a primordial role in the activation and regulation of adaptive immunity. The innate immune system is composed of a cellular arm and a humoral arm. Components of the humoral arm include members of the complement cascade and soluble pattern recognition molecules (PRMs). These fluid-phase PRMs represent the functional ancestors of antibodies and play a crucial role in the discrimination between self, non-self and modified-self. Moreover, evidence has been presented that these soluble PRMs participate in the regulation of inflammatory responses and interact with the cellular arm of the innate immune system. Pentraxins consist of a set of multimeric soluble proteins and represent the prototypic components of humoral innate immunity. Based on the primary structure of the protomer, pentraxins are divided into two groups: short pentraxins and long pentraxins. The short pentraxins C-reactive protein and serum amyloid P-component are produced by the liver and represent the main acute phase proteins in human and mouse, respectively. The long pentraxin PTX3 is produced by innate immunity cells (e.g. PMN, macrophages, dendritic cells), interacts with several ligands and plays an essential role in innate immunity, tuning inflammation and matrix deposition. PTX3 provides a paradigm for the mode of action of humoral innate immunity.     

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), 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 (K(D) = 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.     

The inflammatory protein Pentraxin 3 in cardiovascular disease

The acute phase protein Pentraxin 3 (PTX3) plays a non-redundant role as a soluble pattern recognition receptor for selected pathogens and it represents a rapid biomarker for primary local activation of innate immunity and inflammation. Recent evidence indicates that PTX3 exerts an important role in modulating the cardiovascular system in humans and experimental models. In particular, there are conflicting points concerning the effects of PTX3 in cardiovascular diseases (CVD) since several observations indicate a cardiovascular protective effect of PTX3 while others speculate that the increased plasma levels of PTX3 in subjects with CVD correlate with disease severity and with poor prognosis in elderly patients.In the present review, we discuss the multifaceted effects of PTX3 on the cardiovascular system focusing on its involvement in atherosclerosis, endothelial function, hypertension, myocardial infarction and angiogenesis. This may help to explain how the specific modulation of PTX3 such as the use of different dosing, time, and target organs could help to contain different vascular diseases. These opposite actions of PTX3 will be emphasized concerning the modulation of cardiovascular system where potential therapeutic implications of PTX3 in humans are discussed.     

Acute stressor exposure facilitates innate immunity more in physically active than in sedentary rats

Most previous stress-immune research focused on the immunosuppressive effects of stress on acquired immunity. More recently, it has become clear that acute stressor exposure can potentiate innate, as well as suppress acquired, immunity. For example, acute stress improves recovery from bacterial inflammation, a classic in vivo measure of innate immunity. The previous work was done in sedentary organisms. Physical activity status can modulate the impact of stress on immune function. The following studies tested the hypothesis that the effect of stress on inflammation after subcutaneous challenge with bacteria (Escherichia coli) is facilitated by physical activity. The results were that sedentary, stressed rats resolved their inflammation 1-2 days faster and have increased circulating neutrophils compared with their nonstressed, sedentary counterparts. In contrast, physically active, stressed rats resolve their inflammation 3-4 days faster and have increased circulating and inflammatory site neutrophils compared with their nonstressed counterparts. Importantly, the beneficial impact of stress on inflammation recovery and neutrophil migration was greater in the physically active, than sedentary, stressed rats. Thus physical activity status facilitates the positive effect of acute stress on innate immunity.     

HELP LDL Apheresis Reduces Plasma Pentraxin 3 in Familial Hypercholesterolemia

Background

Pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity, is secreted by vascular cells in response to injury, possibly aiming at tuning arterial activation associated with vascular damage. Severe hypercholesterolemia as in familial hypercholesterolemia (FH) promotes vascular inflammation and atherosclerosis; low-density lipoprotein (LDL) apheresis is currently the treatment of choice to reduce plasma lipids in FH. HELP LDL apheresis affects pro- and antiinflammatory biomarkers, however its effects on PTX3 levels are unknown. We assessed the impact of FH and of LDL removal by HELP apheresis on PTX3.

Methods

Plasma lipids, PTX3, and CRP were measured in 19 patients with FH undergoing chronic HELP LDL apheresis before and after treatment and in 20 control subjects. In the patients assessment of inflammation and oxidative stress markers included also plasma TNFα, fibrinogen and TBARS.

Results

At baseline, FH patients had higher (p = 0.0002) plasma PTX3 than matched control subjects. In FH PTX3 correlated positively (p≤0.05) with age, gender and CRP and negatively (p = 0.01) with HELP LDL apheresis vintage. The latter association was confirmed after correction for age, gender and CRP. HELP LDL apheresis acutely reduced (p≤0.04) plasma PTX3, CRP, fibrinogen, TBARS and lipids, but not TNFα. No association was observed between mean decrease in PTX3 and in LDL cholesterol. PTX3 paralleled lipids, oxidative stress and inflammation markers in time-course study.

Conclusion

FH is associated with increased plasma PTX3, which is acutely reduced by HELP LDL apheresis independently of LDL cholesterol, as reflected by the lack of association between change in PTX3 and in LDL levels. These results, together with the finding of a negative relationship between PTX3 and duration of treatment suggest that HELP LDL apheresis may influence both acutely and chronically cardiovascular outcomes in FH by modulating PTX3.     

M-ficolin interacts with the long pentraxin PTX3: a novel case of cross-talk between soluble pattern-recognition molecules

Ficolins and pentraxins are soluble oligomeric pattern-recognition molecules that sense danger signals from pathogens and altered self-cells and might act synergistically in innate immune defense and maintenance of immune tolerance. The interaction of M-ficolin with the long pentraxin pentraxin 3 (PTX3) has been characterized using surface plasmon resonance spectroscopy and electron microscopy. M-ficolin was shown to bind PTX3 with high affinity in the presence of calcium ions. The interaction was abolished in the presence of EDTA and inhibited by N-acetyl-D-glucosamine, indicating involvement of the fibrinogen-like domain of M-ficolin. Removal of sialic acid from the single N-linked carbohydrate of the C-terminal domain of PTX3 abolished the interaction. Likewise, an M-ficolin mutant with impaired sialic acid-binding ability did not interact with PTX3. Interaction was also impaired when using the isolated recognition domain of M-ficolin or the monomeric C-terminal domain of PTX3, indicating requirement for oligomerization of both proteins. Electron microscopy analysis of the M-ficolin-PTX3 complexes revealed that the M-ficolin tetramer bound up to four PTX3 molecules. From a functional point of view, immobilized PTX3 was able to trigger M-ficolin-dependent activation of the lectin complement pathway. These data indicate that interaction of M-ficolin with PTX3 arises from its ability to bind sialylated ligands and thus differs from the binding to the short pentraxin C-reactive protein and from the binding of L-ficolin to PTX3. The M-ficolin-PTX3 interaction described in this study represents a novel case of cross-talk between soluble pattern-recognition molecules, lending further credit to the integrated view of humoral innate immunity that emerged recently.     

The long pentraxin PTX3 is synthesized in IgA glomerulonephritis and activates mesangial cells

The long pentraxin PTX3 has been recently involved in amplification of the inflammatory reactions and regulation of innate immunity. In the present study we evaluated the expression and role of PTX3 in glomerular inflammation. PTX3 expression was investigated in the IgA, type I membranoproliferative, and diffuse proliferative lupus glomerulonephritis, which are characterized by inflammatory and proliferative lesions mainly driven by resident mesangial cells, and in the membranous glomerulonephritis and the focal segmental glomerular sclerosis, where signs of glomerular inflammation are usually absent. We found an intense staining for PTX3 in the expanded mesangial areas of renal biopsies obtained from patients with IgA glomerulonephritis. The pattern of staining was on glomerular mesangial and endothelial cells. Scattered PTX3-positive cells were also detected in glomeruli of type I membranoproliferative glomerulonephritis. The concomitant expression of CD14 suggests an inflammatory origin of these cells. Normal renal tissue and biopsies from patients with the other glomerular nephropathies studied were mainly negative for PTX3 expression in glomeruli. However, PTX3-positive cells were detected in the interstitium of nephropathies showing inflammatory interstitial injury. In vitro, cultured human mesangial cells synthesized PTX3 when stimulated with TNF-alpha and IgA and exhibited specific binding for recombinant PTX3. Moreover, stimulation with exogenous PTX3 promoted mesangial cell contraction and synthesis of the proinflammatory lipid mediator platelet-activating factor. In conclusion, we provide the first evidence that mesangial cells may both produce and be a target for PTX3. The detection of this long pentraxin in the renal tissue of patients with glomerulonephritis suggests its potential role in the modulation of glomerular and tubular injury.     

Immunity in plants and animals: common ends through different means using similar tools

A comparative approach is potentially useful for understanding the role of mammal innate immunity role in stimulating adaptive immunity as well as the relationship between these two types of immune strategies. Considerable progress has been made in the elucidation of the co-ordinated events involved in plant perception of infection and their mobilisation of defence responses. Although lacking immunoglobulin molecules, circulating cells, and phagocytic processes, plants successfully use pre-formed physical and chemical innate defences, as well as inducible adaptive immune strategies. In the present paper, we review some shared and divergent immune aspects present in both animals and plants.     

Heterocomplexes of mannose-binding lectin and the pentraxins PTX3 or serum amyloid P component trigger cross-activation of the complement system

The long pentraxin 3 (PTX3), serum amyloid P component (SAP), and C-reactive protein belong to the pentraxin family of pattern recognition molecules involved in tissue homeostasis and innate immunity. They interact with C1q from the classical complement pathway. Whether this also occurs via the analogous mannose-binding lectin (MBL) from the lectin complement pathway is unknown. Thus, we investigated the possible interaction between MBL and the pentraxins. We report that MBL bound PTX3 and SAP partly via its collagen-like domain but not C-reactive protein. MBL-PTX3 complex formation resulted in recruitment of C1q, but this was not seen for the MBL-SAP complex. However, both MBL-PTX3 and MBL-SAP complexes enhanced C4 and C3 deposition and opsonophagocytosis of Candida albicans by polymorphonuclear leukocytes. Interaction between MBL and PTX3 led to communication between the lectin and classical complement pathways via recruitment of C1q, whereas SAP-enhanced complement activation occurs via a hitherto unknown mechanism. Taken together, MBL-pentraxin heterocomplexes trigger cross-activation of the complement system.     

High-volume ventilation induces pentraxin 3 expression in multiple acute lung injury models in rats

Pentraxin 3 (PTX3) is an acute-phase protein, which can be produced by a variety of tissue cells at the site of infection or inflammation. It plays an important role in innate immunity in the lung and in mediating acute lung injury. The aim of this study was to determine the effect of mechanical ventilation on PTX3 expression in multiple lung injury models. Male Sprague-Dawley rats were challenged with intravenous injection of lipopolysaccharide (LPS) or hemorrhage followed by resuscitation (HS). The animals were then subjected to either relatively higher (12 ml/kg) or lower (6 ml/kg, positive end-expiratory pressure of 5 cmH(2)O) volume ventilation for 4 h. High-volume ventilation significantly enhanced PTX3 expression in the lung, either alone or in combination with LPS or hemorrhage. A significant increase of PTX3 immunohistochemistry staining in the lung was seen in all injury groups. The PTX3 expression was highly correlated with the severity of lung injury determined by blood gas, lung elastance, and wet-to-dry ratio. To determine the effects of HS, LPS, or injurious ventilation (25 ml/kg) alone on PTX3 expression, another group of rats was studied. Injurious ventilation significantly damaged the lung and increased PTX3 expression. A local expression of PTX3 induced by high-volume ventilation, either alone or in combination with other pathological conditions, suggests that it may be an important mediator in ventilator-induced lung injury.     

Long pentraxin 3: A novel multifaceted player in cancer

Since its discovery in 1992, long pentraxin 3 (PTX3) has been characterized as soluble patter recognition receptor, a key player of the innate immunity arm with non-redundant functions in pathogen recognition and inflammatory responses. As a component of the extra-cellular matrix milieu, PTX3 has been implicated also in wound healing/tissue remodeling, cardiovascular diseases, fertility, and infectious diseases. Consequently, PTX3 levels in biological fluids have been proposed as a fluid-phase biomarker in different pathological conditions.In the last decade, experimental evidences have shown that PTX3 may exert a significant impact also on different aspects of cancer biology, including tumor onset, angiogenesis, metastatic dissemination and immune-modulation. However, it remains unclear whether PTX3 acts as a good cop or bad cop in cancer. In this review, we will summarize and discuss the scientific literature data focusing on the role of PTX3 in experimental and human tumors, including its putative translational implications.     

Multidisciplinary perspectives on mechanisms of activity of popular immune-enhancing herbal supplements used by athletes

This paper examines cellular and molecular mechanisms that may underpin the purported effects of five herbal supplements in the context of athlete immune function. Ginseng and echinacea are used frequently by athletes, whereas astragalus and elderberry are used infrequently and pequi is just emerging as a possible supplement. In vivo studies of these products on athlete immune function have yielded heterogeneous results, likely due to experimental design differences. Ginseng, echinacea, elderberry, and pequi are considered asterids sensu lato. Ginseng appears to exert strongest effects on components of adaptive immunity, in particular maintaining Th1/Th2 balance of CD4⁺T cells and their downstream effects, via its ginsenosides, flavonoids, and polysaccharides. Echinacea alkamides, caffeic acid derivatives, and polysacchardies may target both innate and adaptive immunity, though perhaps the former more consistently. Elderberry harbors anthocyanins and lectins which may modulate innate immunity. Data on pequi is limited but suggests that carotenoids, phenols, and fatty acids may alter circulating leukocyte populations. More phylogenetically distant, astragalus is a rosid sensu lato and may influence the innate immune system through flavonoids, polysaccharides, and saponins. Supplements generally demonstrate no effects on physiologic parameters such as lactate, oxygen dynamics, or athletic performance. Bioavailability studies indicate that purported bioactive molecules of these supplements may reach circulation in low but therapeutically-relevant quantities. Difficulties in crosscomparisons due to study design differences, coupled with an overall dearth of research on the topic, currently hamper any formal conclusions regarding the efficacy of these supplements as immunoregulators for athletes.     

Identification of an antiangiogenic FGF2-binding site in the N terminus of the soluble pattern recognition receptor PTX3

Long-pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 comprises a pentraxin-like C-terminal domain involved in complement activation via C1q interaction and an N-terminal extension with unknown functions. PTX3 binds fibroblast growth factor-2 (FGF2), inhibiting its pro-angiogenic and pro-restenotic activity. Here, retroviral transduced endothelial cells (ECs) overexpressing the N-terminal fragment PTX3-(1-178) showed reduced mitogenic activity in response to FGF2. Accordingly, purified recombinant PTX3-(1-178) binds FGF2, prevents PTX3/FGF2 interaction, and inhibits FGF2 mitogenic activity in ECs. Also, the monoclonal antibody mAb-MNB4, which recognizes the PTX3-(87-99) epitope, prevents FGF2/PTX3 interaction and abolishes the FGF2 antagonist activity of PTX3. Consistently, the synthetic peptides PTX3-(82-110) and PTX3-(97-110) bind FGF2 and inhibit the interaction of FGF2 with PTX3 immobilized to a BIAcore sensor chip, FGF2-dependent EC proliferation, and angiogenesis in vivo. Thus, the data identify a FGF2-binding domain in the N-terminal extension of PTX3 spanning the PTX3-(97-110) region, pointing to a novel function for the N-terminal extension of PTX3 and underlining the complexity of the PTX3 molecule for modular humoral pattern recognition.     

Toll-like receptors, inflammation, metabolism and obesity

Obesity is a highly prevalent health problem in Western countries that leads to many important diseases such as type 2 diabetes and metabolic syndrome being now considered an inflammatory chronic disease. Adipocytes are no longer considered passive cells storing fat since they are major producers of inflammatory cytokines during obesity. Adipocytes and macrophages share many biological properties including the synthesis of similar molecules regulating inflammation. Fatty acid levels are elevated in obesity and induce inflammatory pathways by yet a mostly unknown mechanism, leading to the development of insulin and leptin resistance. Recent studies suggest that these effects could be mediated through the activation of toll-like receptors (TLR). TLR signalling pathways might contribute to the development of obesity-associated insulin resistance, thus representing a connection between innate immunity and metabolism. Here, we summarize the recent evidence for the important role that TLRs play in adipose tissue, obesity and insulin resistance.     

Structure and function of the long pentraxin PTX3 glycosidic moiety: fine-tuning of the interaction with C1q and complement activation

The prototypic long pentraxin PTX3 is a unique fluid-phase pattern recognition receptor that plays a nonredundant role in innate immunity and female fertility. The PTX3 C-terminal domain is required for C1q recognition and complement activation and contains a single N-glycosylation site on Asn 220. In the present study, we characterized the structure of the human PTX3 glycosidic moiety and investigated its relevance in C1q interaction and activation of the complement classical pathway. By specific endo and exoglycosidases digestion and direct mass spectrometric analysis, we found that both recombinant and naturally occurring PTX3 were N-linked to fucosylated and sialylated complex-type sugars. Interestingly, glycans showed heterogeneity mainly in the relative amount of bi, tri, and tetrantennary structures depending on the cell type and inflammatory stimulus. Enzymatic removal of sialic acid or the entire glycosidic moiety equally enhanced PTX3 binding to C1q compared to that in the native protein, thus indicating that glycosylation substantially contributes to modulate PTX3/C1q interaction and that sialic acid is the main determinant of this contribution. BIAcore kinetic measurements returned decreasing K(off) values as sugars were removed, pointing to a stabilization of the PTX3/C1q complex. No major rearrangement of PTX3 quaternary structure was observed after desialylation or deglycosylation as established by size exclusion chromatography. Consistent with C1q binding, PTX3 desialylation enhanced the activation of the classical complement pathway, as assessed by C4 and C3 deposition. In conclusion, our results provided evidence of an involvement of the PTX3 sugar moiety in C1q recognition and complement activation.     

Human pentraxin 3 binds to the complement regulator c4b-binding protein

The long pentraxin 3 (PTX3) is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP). A PTX3-binding site was identified within short consensus repeats 1-3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage.