PTX3, a Humoral Pattern Recognition Molecule, in Innate Immunity, Tissue Repair, and Cancer.

Innate immunity includes a cellular and a humoral arm. PTX3 is a fluid-phase pattern recognition molecule conserved in evolution which acts as a key component of humoral innate immunity in infections of fungal, bacterial, and viral origin. PTX3 binds conserved microbial structures and self-components under conditions of inflammation and activates effector functions (complement, phagocytosis). Moreover, it has a complex regulatory role in inflammation, such as ischemia/reperfusion injury and cancer-related inflammation, as well as in extracellular matrix organization and remodeling, with profound implications in physiology and pathology. Finally, PTX3 acts as an extrinsic oncosuppressor gene by taming tumor-promoting inflammation in murine and selected human tumors. Thus evidence suggests that PTX3 is a key homeostatic component at the crossroad of innate immunity, inflammation, tissue repair, and cancer. Dissecting the complexity of PTX3 pathophysiology and human genetics paves the way to diagnostic and therapeutic exploitation.

Genetic Deficiency of the Long Pentraxin 3 Affects Osteogenesis and Osteoclastogenesis in Homeostatic and Inflammatory Conditions.

The long pentraxin 3 (PTX3) is a soluble glycoprotein made by immune and nonimmune cells endowed with pleiotropic functions in innate immunity, inflammation, and tissue remodeling. PTX3 has recently emerged as a mediator of bone turnover in both physiological and pathological conditions, with direct and indirect effects on osteoblasts and osteoclasts. This notwithstanding, its role in bone biology, with major regard to the osteogenic potential of osteoblasts and their interplay with osteoclasts, is at present unclear. Here, we investigated the contribution of this pentraxin to bone deposition in the osteogenic lineage by assessing collagen production, mineralization capacity, osteoblast maturation, extracellular matrix gene expression, and inflammatory mediators' production in primary osteoblasts from the calvaria of wild-type (WT) and Ptx3-deficient (Ptx3-/-) mice. Also, we evaluated the effect of PTX3 on osteoclastogenesis in cocultures of primary osteoblasts and bone marrow-derived osteoclasts. Our investigations were carried out both in physiological and inflammatory conditions to recapitulate in vitro aspects of inflammatory diseases of the bone. We found that primary osteoblasts from WT animals constitutively expressed low levels of the protein in osteogenic noninflammatory conditions, and genetic ablation of PTX3 in these cells had no major impact on collagen and hydroxyapatite deposition. However, Ptx3-/- osteoblasts had an increased RANKL/OPG ratio and CD44 expression, which resulted in in enhanced osteoclastogenesis when cocultured with bone marrow monocytes. Inflammation (modelled through administration of tumor necrosis factor-α, TNF-α) boosted the expression and accumulation of PTX3 and inflammatory mediators in WT osteoblasts. In these conditions, Ptx3 genetic depletion was associated with reduced collagen deposition and immune modulators' production. Our study shed light on the role of PTX3 in osteoblast and osteoclast biology and identified a major effect of inflammation on the bone-related properties of this pentraxin, which might be relevant for therapeutic and/or diagnostic purposes in musculoskeletal pathology.

Where Are We with RPE Replacement Therapy? A Translational Review from the Ophthalmologist Perspective.

The retinal pigmented epithelium (RPE) plays a pivotal role in retinal homeostasis. It is therefore an interesting target to fill the unmet medical need of different retinal diseases, including age-related macular degeneration and Stargardt disease. RPE replacement therapy may use different cellular sources: induced pluripotent stem cells or embryonic stem cells. Cells can be transferred as suspension on a patch with different surgical approaches. Results are promising although based on very limited samples. In this review, we summarize the current progress of RPE replacement and provide a comparative assessment of different published approaches which may become standard of care in the future.

Genetic Aspects of Age-Related Macular Degeneration and Their Therapeutic Potential.

Age-related macular degeneration (AMD) is a complex and multifactorial disease, resulting from the interaction of environmental and genetic factors. The continuous discovery of associations between genetic polymorphisms and AMD gives reason for the pivotal role attributed to the genetic component to its development. In that light, genetic tests and polygenic scores have been created to predict the risk of development and response to therapy. Still, none of them have yet been validated. Furthermore, there is no evidence from a clinical trial that the determination of the individual genetic structure can improve treatment outcomes. In this comprehensive review, we summarize the polymorphisms of the main pathogenetic ways involved in AMD development to identify which of them constitutes a potential therapeutic target. As complement overactivation plays a major role, the modulation of targeted complement proteins seems to be a promising therapeutic approach. Herein, we summarize the complement-modulating molecules now undergoing clinical trials, enlightening those in an advanced phase of trial. Gene therapy is a potential innovative one-time treatment, and its relevance is quickly evolving in the field of retinal diseases. We describe the state of the art of gene therapies now undergoing clinical trials both in the field of complement-suppressors and that of anti-VEGF.

TNF-Stimulated Gene-6 Is a Key Regulator in Switching Stemness and Biological Properties of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well established to have promising therapeutic properties. TNF-stimulated gene-6 (TSG-6), a potent tissue-protective and anti-inflammatory factor, has been demonstrated to be responsible for a significant part of the tissue-protecting properties mediated by MSCs. Nevertheless, current knowledge about the biological function of TSG-6 in MSCs is limited. Here, we demonstrated that TSG-6 is a crucial factor that influences many functional properties of MSCs. The transcriptomic sequencing analysis of wild-type (WT) and TSG-6-/- -MSCs shows that the loss of TSG-6 expression leads to the perturbation of several transcription factors, cytokines, and other key biological pathways. TSG-6-/- -MSCs appeared morphologically different with dissimilar cytoskeleton organization, significantly reduced size of extracellular vesicles, decreased cell proliferative rate, and loss of differentiation abilities compared with the WT cells. These cellular effects may be due to TSG-6-mediated changes in the extracellular matrix (ECM) environment. The supplementation of ECM with exogenous TSG-6, in fact, rescued cell proliferation and changes in morphology. Importantly, TSG-6-deficient MSCs displayed an increased capacity to release interleukin-6 conferring pro-inflammatory and pro-tumorigenic properties to the MSCs. Overall, our data provide strong evidence that TSG-6 is crucial for the maintenance of stemness and other biological properties of murine MSCs.

Pentraxins in the activation and regulation of innate immunity.

Humoral fluid phase pattern recognition molecules (PRMs) are a key component of the activation and regulation of innate immunity. Humoral PRMs are diverse. We focused on the long pentraxin PTX3 as a paradigmatic example of fluid phase PRMs. PTX3 acts as a functional ancestor of antibodies and plays a non-redundant role in resistance against selected microbes in mouse and man and in the regulation of inflammation. This molecule interacts with complement components, thus modulating complement activation. In particular, PTX3 regulates complement-driven macrophage-mediated tumor progression, acting as an extrinsic oncosuppressor in preclinical models and selected human tumors. Evidence collected over the years suggests that PTX3 is a biomarker and potential therapeutic agent in humans, and pave the way to translation of this molecule into the clinic.

Complement factor H in host defense and immune evasion.

Complement is the major humoral component of the innate immune system. It recognizes pathogen- and damage-associated molecular patterns, and initiates the immune response in coordination with innate and adaptive immunity. When activated, the complement system unleashes powerful cytotoxic and inflammatory mechanisms, and thus its tight control is crucial to prevent damage to host tissues and allow restoration of immune homeostasis. Factor H is the major soluble inhibitor of complement, where its binding to self markers (i.e., particular glycan structures) prevents complement activation and amplification on host surfaces. Not surprisingly, mutations and polymorphisms that affect recognition of self by factor H are associated with diseases of complement dysregulation, such as age-related macular degeneration and atypical haemolytic uremic syndrome. In addition, pathogens (i.e., non-self) and cancer cells (i.e., altered-self) can hijack factor H to evade the immune response. Here we review recent (and not so recent) literature on the structure and function of factor H, including the emerging roles of this protein in the pathophysiology of infectious diseases and cancer.

Genetic PTX3 deficiency and aspergillosis in stem-cell transplantation.

BACKGROUND: The soluble pattern-recognition receptor known as long pentraxin 3 (PTX3) has a nonredundant role in antifungal immunity. The contribution of single-nucleotide polymorphisms (SNPs) in PTX3 to the development of invasive aspergillosis is unknown. METHODS: We screened an initial cohort of 268 patients undergoing hematopoietic stem-cell transplantation (HSCT) and their donors for PTX3 SNPs modifying the risk of invasive aspergillosis. The analysis was also performed in a multicenter study involving 107 patients with invasive aspergillosis and 223 matched controls. The functional consequences of PTX3 SNPs were investigated in vitro and in lung specimens from transplant recipients. RESULTS: Receipt of a transplant from a donor with a homozygous haplotype (h2/h2) in PTX3 was associated with an increased risk of infection, in both the discovery study (cumulative incidence, 37% vs. 15%; adjusted hazard ratio, 3.08; P=0.003) and the confirmation study (adjusted odds ratio, 2.78; P=0.03), as well as with defective expression of PTX3. Functionally, PTX3 deficiency in h2/h2 neutrophils, presumably due to messenger RNA instability, led to impaired phagocytosis and clearance of the fungus. CONCLUSIONS: Genetic deficiency of PTX3 affects the antifungal capacity of neutrophils and may contribute to the risk of invasive aspergillosis in patients treated with HSCT. (Funded by the European Society of Clinical Microbiology and Infectious Diseases and others.).

PTX3 as a paradigm for the interaction of pentraxins with the complement system.

Pentraxins are highly conserved components of the humoral arm of innate immunity. They include the short pentraxins C reactive protein (CRP) and serum amyloid P component (SAP), and the long pentraxin PTX3. These are soluble pattern-recognition molecules that are present in the blood and body fluids, and share the ability to recognize pathogens and promote their disposal. CRP and SAP are produced systemically in the liver while PTX3 is produced locally in a number of tissues, macrophages and neutrophils being major sources of this long pentraxin. Pentraxins interact with components of the classical and lectin pathways of Complement as well as with Complement regulators. In particular, PTX3 recognizes C1q, factor H, MBL and ficolins, where these interactions amplify the repertoire of microbial recognition and effector functions of the Complement system. The complex interaction of pentraxins with the Complement system at different levels has broad implications for host defence and regulation of inflammation.

The pentraxins PTX3 and SAP in innate immunity, regulation of inflammation and tissue remodelling.

Pentraxins are a superfamily of fluid phase pattern recognition molecules conserved in evolution and characterized by a cyclic multimeric structure. C-reactive protein (CRP) and serum amyloid P component (SAP) constitute the short pentraxin arm of the superfamily. CRP and SAP are produced in the liver in response to IL-6 and are acute phase reactants in humans and mice respectively. In addition SAP has been shown to affect tissue remodelling and fibrosis by stabilizing all types of amyloid fibrils and by regulating monocyte to fibrocyte differentiation. Pentraxin 3 (PTX3) is the prototype of the long pentraxin arm. Gene targeted mice and genetic and epigenetic studies in humans suggest that PTX3 plays essential non-redundant roles in innate immunity and inflammation as well as in tissue remodelling. Recent studies have revealed the role of PTX3 as extrinsic oncosuppressor, able to tune cancer-related inflammation. In addition, at acidic pH PTX3 can interact with provisional matrix components promoting inflammatory matrix remodelling. Thus acidification during tissue repair sets PTX3 in a tissue remodelling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity.

PTX3 binds MD-2 and promotes TRIF-dependent immune protection in aspergillosis.

The long pentraxin 3 (PTX3) modulates different effector pathways involved in innate resistance to Aspergillus fumigatus, including complement activation or promotion of phagocytosis by interacting with FcγRs. However, whether and how TLRs modulate PTX3 mediates antifungal resistance is not known. In this study, we demonstrate that PTX3 binds myeloid differentiation protein 2 (MD-2) in vitro and exerts its protective antifungal activity in vivo through TLR4/MD-2-mediated signaling. Similar to Tlr4(-/-) mice, Md2(-/-) mice displayed high susceptibility to pulmonary aspergillosis, a phenotype associated with a proinflammatory cytokine profile and impaired antifungal activity of polymorphonuclear neutrophils. Treating Md2(-/-) mice with PTX3 failed to confer immune protection against the fungus, whereas adoptive transfer of MD-2-competent polymorphonuclear neutrophils restored it. Mechanistically, engagement of MD-2 by PTX3-opsonized Aspergillus conidia activated the TLR4/Toll/IL-1R domain-containing adapter inducing IFN-ß-dependent signaling pathway converging on IL-10. Thus, we have identified a novel receptor mechanism, involving the TLR4/MD-2/Toll/IL-1R domain-containing adapter inducing IFN-ß-mediated signaling, whereby PTX3 elicits antifungal resistance with limited immunopathology in A. fumigatus infection.

Loss of function of Ribonuclease T2, an ancient and phylogenetically conserved RNase, plays a crucial role in ovarian tumorigenesis.

In recent years, the role played by the stromal microenvironment has been given growing attention in order to achieve a full understanding of cancer initiation and progression. Because cancer is a tissue-based disease, the integrity of tissue architecture is a major constraint toward cancer growth. Indeed, a large contribution of the natural resistance to cancer stems from stromal microenvironment components, the dysregulation of which can facilitate cancer occurrence. For instance, recent experimental evidence has highlighted the involvement of stromal cells in ovarian carcinogenesis, as epitomized by ovarian xenografts obtained by a double KO of the murine Dicer and Pten genes. Likewise, we reported the role of an ancient extracellular RNase, called Ribonuclease T2 (RNASET2), within the ovarian stromal microenvironment. Indeed, hyperexpression of RNASET2 is able to control tumorigenesis by recruiting macrophages (mostly of the anticancer M1 subtype) at the tumor sites. We present biological data obtained by RNASET2 silencing in the poorly tumorigenetic and highly RNASET2-expressing human OVCAR3 cell line. RNASET2 knockdown was shown to stimulate in vivo tumor growth early after microinjection of OVCAR3 cells in nude mice. Moreover, we have investigated by molecular profiling the in vivo expression signature of human and mouse cell xenografts and disclosed the activation of pathways related to activation of the innate immune response and modulation of ECM components. Finally, we provide evidence for a role of RNASET2 in triggering an in vitro chemotactic response in macrophages. These results further highlight the critical role played by the microenvironment in RNASET2-mediated ovarian tumor suppression, which could eventually contribute to better clarify the pathogenesis of this disease.

Incorporation of pentraxin 3 into hyaluronan matrices is tightly regulated and promotes matrix cross-linking.

Mammalian oocytes are surrounded by a highly hydrated hyaluronan (HA)-rich extracellular matrix with embedded cumulus cells, forming the cumulus cell·oocyte complex (COC) matrix. The correct assembly, stability, and mechanical properties of this matrix, which are crucial for successful ovulation, transport of the COC to the oviduct, and its fertilization, depend on the interaction between HA and specific HA-organizing proteins. Although the proteins inter-α-inhibitor (IαI), pentraxin 3 (PTX3), and TNF-stimulated gene-6 (TSG-6) have been identified as being critical for COC matrix formation, its supramolecular organization and the molecular mechanism of COC matrix stabilization remain unknown. Here we used films of end-grafted HA as a model system to investigate the molecular interactions involved in the formation and stabilization of HA matrices containing TSG-6, IαI, and PTX3. We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6. This long pentraxin also failed to bind to products of the interaction between IαI, TSG-6, and HA, among which are the covalent heavy chain (HC)·HA and HC·TSG-6 complexes, despite the fact that both IαI and TSG-6 are ligands of PTX3. Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films. Moreover, we demonstrated that this ternary protein mixture made of IαI, PTX3, and TSG-6 is sufficient to promote formation of a stable (i.e. cross-linked) yet highly hydrated HA matrix. We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking.

Interleukin-1ß Polymorphisms Are Genetic Markers of Susceptibility to Periprosthetic Joint Infection in Total Hip and Knee Arthroplasty.

Periprosthetic joint infections (PJIs) are serious complications of prosthetic surgery. The criteria for the diagnosis of PJI integrate clinical and laboratory findings in a complex and sometimes inconclusive workflow. Host immune factors hold potential as diagnostic biomarkers in bone and joint infections. We reported that the humoral pattern-recognition molecule long pentraxin 3 (PTX3) predicts PJI in total hip and knee arthroplasty (THA and TKA, respectively). If and how genetic variation in PTX3 and inflammatory genes that affect its expression (IL-1ß, IL-6, IL-10, and IL-17A) contributes to the risk of PJI is unknown. We conducted a case-control study on a Caucasian historic cohort of THA and TKA patients who had prosthesis explant due to PJI (cases) or aseptic complications (controls). Saliva was collected from 93 subjects and used to extract DNA and genotype PTX3, IL-1ß, IL-6, IL-10, and IL-17A single-nucleotide polymorphisms (SNPs). Moreover, the concentration of IL-1ß, IL-10, and IL-6 was measured in synovial fluid and plasma. No association was found between PTX3 polymorphisms and PJI; however, the AGG haplotype, encompassing rs2853550, rs1143634, and rs1143627 in IL-1ß, was linked to the infection (p = 0.017). Also, synovial levels of all inflammatory markers were higher in cases than in controls, and a correlation emerged between synovial concentration of PTX3 and that of IL-1ß in cases only (Spearman r = 0.67, p = 0.004). We identified a relationship between rs2853550 and the synovial concentration of IL-1ß and PTX3. Our findings suggest that IL-1ß SNPs could be used for the early identification of THA and TKA patients with a high risk of infection.

Long pentraxin 3/tumor necrosis factor-stimulated gene-6 interaction: a biological rheostat for fibroblast growth factor 2-mediated angiogenesis.

OBJECTIVE: Angiogenesis is regulated by the balance between pro- and antiangiogenic factors and by extracellular matrix protein interactions. Fibroblast growth factor 2 (FGF2) is a major proangiogenic inducer inhibited by the interaction with the soluble pattern recognition receptor long pentraxin 3 (PTX3). PTX3 is locally coexpressed with its ligand tumor necrosis factor-stimulated gene-6 (TSG-6), a secreted glycoprotein that cooperates with PTX3 in extracellular matrix assembly. Here, we characterized the effect of TSG-6 on PTX3/FGF2 interaction and FGF2-mediated angiogenesis. METHODS AND RESULTS: Solid phase binding and surface plasmon resonance assays show that TSG-6 and FGF2 bind the PTX3 N-terminal domain with similar affinity. Accordingly, TSG-6 prevents FGF2/PTX3 interaction and suppresses the inhibition exerted by PTX3 on heparan sulfate proteoglycan/FGF2/FGF receptor complex formation and on FGF2-dependent angiogenesis in vitro and in vivo. Also, endogenous PTX3 exerts an inhibitory effect on vascularization induced by FGF2 in a murine subcutaneous Matrigel plug assay, the inhibition being abolished in Ptx3-null mice or by TSG-6 treatment in wild-type animals. CONCLUSION: TSG-6 reverts the inhibitory effects exerted by PTX3 on FGF2-mediated angiogenesis through competition of FGF2/PTX3 interaction. This may provide a novel mechanism to control angiogenesis in those pathological settings characterized by the coexpression of TSG-6 and PTX3, in which the relative levels of these proteins may fine-tune the angiogenic activity of FGF2.

Structural insights into the biological functions of the long pentraxin PTX3.

Soluble pattern recognition molecules (PRMs) are a heterogenous group of proteins that recognize pathogen- and danger-associated molecular patterns (PAMPs and DAMPs, respectively), and cooperate with cell-borne receptors in the orchestration of innate and adaptive immune responses to pathogenic insults and tissue damage. Amongst soluble PRMs, pentraxins are a family of highly conserved proteins with distinctive structural features. Originally identified in the early 1990s as an early inflammatory gene, PTX3 is the prototype of long pentraxins. Unlike the short pentraxin C reactive protein (CRP), whose expression is mostly confined to the liver, PTX3 is made by several immune and non-immune cells at sites of infection and inflammation, where it intercepts fundamental aspects of infection immunity, inflammation, and tissue remodeling. Of note, PTX3 cross talks to components of the complement system to control cancer-related inflammation and disposal of pathogens. Also, it is an essential component of inflammatory extracellular matrices (ECMs) through crosslinking of hyaluronic acid and turn-over of provisional fibrin networks that assemble at sites of tissue injury. This functional diversity is mediated by unique structural characteristics whose fine details have been unveiled only recently. Here, we revisit the structure/function relationships of this long pentraxin in light of the most recent advances in its structural biology, with a focus on the interplay with complement and the emerging roles as a component of the ECM. Differences to and similarities with the short pentraxins are highlighted and discussed.

Long Pentraxin 3 as a New Biomarker for Diagnosis of Hip and Knee Periprosthetic Joint Infections.

BACKGROUND: Preoperative diagnosis of periprosthetic joint infections (PJIs) poses an unmet clinical challenge. The long pentraxin PTX3 is a component of the innate immune system involved in infection immunity. This study evaluated the potential of synovial and plasmatic PTX3 in the diagnosis of hip and knee PJIs. METHODS: Consecutive total hip and knee arthroplasty (THA/TKA) revisions were prospectively included and classified as septic or aseptic according to the European Bone and Joint Infection Society (EBJIS) and Musculoskeletal Infection Society (MSIS) criteria. The concentration of PTX3 in plasma and synovial fluid samples was measured with ELISA. The AUC, threshold value, sensitivity, specificity, and positive and negative likelihood ratios were calculated using the ROC (receiver operating characteristic) curve method. RESULTS: The study population included 128 patients (94 THAs; 34 TKAs). The AUC of the synovial PTX3 based on EBJIS criteria was 0.85 (p < 0.0001), with a sensitivity of 81.13% and a specificity of 93.33%. The AUC based on MSIS criteria was 0.95 (p < 0.001), with a sensitivity of 91.43% and a specificity of 89.25%. Plasmatic PTX3 failed to discriminate infected from non-infected patients. CONCLUSIONS: Synovial PTX3 demonstrated an excellent diagnostic potential in hip and knee PJIs, with a very high specificity irrespective of the diagnostic criteria for PJI.

Regulation of inflammation and protection against invasive pneumococcal infection by the long pentraxin PTX3.

Streptococcus pneumoniae is a major pathogen in children, elderly subjects, and immunodeficient patients. Pentraxin 3 (PTX3) is a fluid-phase pattern recognition molecule (PRM) involved in resistance to selected microbial agents and in regulation of inflammation. The present study was designed to assess the role of PTX3 in invasive pneumococcal infection. In a murine model of invasive pneumococcal infection, PTX3 was strongly induced in non-hematopoietic (particularly, endothelial) cells. The IL-1ß/MyD88 axis played a major role in regulation of the Ptx3 gene expression. Ptx3-/- mice presented more severe invasive pneumococcal infection. Although high concentrations of PTX3 had opsonic activity in vitro, no evidence of PTX3-enhanced phagocytosis was obtained in vivo. In contrast, Ptx3-deficient mice showed enhanced recruitment of neutrophils and inflammation. Using P-selectin-deficient mice, we found that protection against pneumococcus was dependent upon PTX3-mediated regulation of neutrophil inflammation. In humans, PTX3 gene polymorphisms were associated with invasive pneumococcal infections. Thus, this fluid-phase PRM plays an important role in tuning inflammation and resistance against invasive pneumococcal infection.

Human C-type lectin domain family 4, member C (CLEC4C/BDCA-2/CD303) is a receptor for asialo-galactosyl-oligosaccharides.

Plasmacytoid dendritic cells are specialized in the production of type I interferon (type I IFN), which promotes antiviral and antitumor responses, as well as autoimmune disorders. Activation of type I IFN secretion depends on the pattern recognition receptors TLR7 and TLR9, which sense microbial RNA and DNA, respectively. Type I IFN production is modulated by several receptors, including the type II C-type lectin domain family 4, member C (CLEC4C). The natural ligand of CLEC4C is unknown. To identify it, here we probed a glycan array with a soluble form of the CLEC4C ectodomain. We found that CLEC4C recognizes complex type sugars with terminal galactose. Importantly, soluble CLEC4C bound peripheral blood leukocytes and tumor cells that express glycans with galactose residues at the non-reducing ends. The positive and negative modulation of galactose residues on cell membranes was paralleled by the regulation of type I IFN secretion by plasmacytoid dendritic cells in co-culture experiments in vitro. These results suggest that the modulation in the expression of non-sialylated oligosaccharides by invading pathogens or transformed cells may affect type I IFN response and immune surveillance.