The long Pentraxin PTX3 serves as an early predictive biomarker of co-infections in COVID-19.

BACKGROUND: COVID-19 clinical course is highly variable and secondary infections contribute to COVID-19 complexity. Early detection of secondary infections is clinically relevant for patient outcome. Procalcitonin (PCT) and C-reactive protein (CRP) are the most used biomarkers of infections. Pentraxin 3 (PTX3) is an acute phase protein with promising performance as early biomarker in infections. In patients with COVID-19, PTX3 plasma concentrations at hospital admission are independent predictor of poor outcome. In this study, we assessed whether PTX3 contributes to early identification of co-infections during the course of COVID-19. METHODS: We analyzed PTX3 levels in patients affected by COVID-19 with (n = 101) or without (n = 179) community or hospital-acquired fungal or bacterial secondary infections (CAIs or HAIs). FINDINGS: PTX3 plasma concentrations at diagnosis of CAI or HAI were significantly higher than those in patients without secondary infections. Compared to PCT and CRP, the increase of PTX3 plasma levels was associated with the highest hazard ratio for CAIs and HAIs (aHR 11.68 and 24.90). In multivariable Cox regression analysis, PTX3 was also the most significant predictor of 28-days mortality or intensive care unit admission of patients with potential co-infections, faring more pronounced than CRP and PCT. INTERPRETATION: PTX3 is a promising predictive biomarker for early identification and risk stratification of patients with COVID-19 and co-infections. FUNDING: Dolce & Gabbana fashion house donation; Ministero della Salute for COVID-19; EU funding within the MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT) and MUR PNRR Italian network of excellence for advanced diagnosis (Project no. PNC-E3-2022-23683266 PNC-HLS-DA); EU MSCA (project CORVOS 860044).

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.

Polymeric nanocapsules loaded with poly(I:C) and resiquimod to reprogram tumor-associated macrophages for the treatment of solid tumors.

Background: In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer. Toll-like receptors (TLRs) ligands, such as poly(I:C) or resiquimod (R848) are able to reprogram TAMs towards M1-like antitumor effector cells. The objective of our work has been to develop and evaluate polymeric nanocapsules (NCs) loaded with poly(I:C)+R848, to improve drug stability and systemic toxicity, and evaluate their targeting and therapeutic activity towards TAMs in the TME of solid tumors. Methods: NCs were developed by the solvent displacement and layer-by-layer methodologies and characterized by dynamic light scattering and nanoparticle tracking analysis. Hyaluronic acid (HA) was chemically functionalized with mannose for the coating of the NCs to target TAMs. NCs loaded with TLR ligands were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry, using primary human monocyte-derived macrophages. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry and multispectral immunophenotyping. Results: We have developed polymeric NCs loaded with poly(I:C)+R848. Among a series of 5 lead prototypes, protamine-NCs were selected based on their physicochemical properties (size, charge, stability) and in vitro characterization, showing good biocompatibility on primary macrophages and ability to stimulate their production of T-cell attracting chemokines (CXCL10, CCL5) and to induce M1-like macrophages cytotoxicity towards tumor cells. In mouse tumor models, the intratumoral injection of poly(I:C)+R848-protamine-NCs significantly prevented tumor growth and lung metastasis. In an orthotopic murine lung cancer model, the intravenous administration of poly(I:C)+R848-prot-NCs, coated with an additional layer of HA-mannose to improve TAM-targeting, resulted in good antitumoral efficacy with no apparent systemic toxicity. While no significant alterations were observed in T cell numbers (CD8, CD4 or Treg), TAM-reprogramming in treated mice was confirmed by the relative decrease of interstitial versus alveolar macrophages, having higher CD86 expression but lower CD206 and Arg1 expression in the same cells, in treated mice. Conclusion: Mannose-HA-protamine-NCs loaded with poly(I:C)+R848 successfully reprogram TAMs in vivo, and reduce tumor progression and metastasis spread in mouse tumors.

A cytokine/PTX3 prognostic index as a predictor of mortality in sepsis.

Background: Early prognostic stratification of patients with sepsis is a difficult clinical challenge. Aim of this study was to evaluate novel molecules in association with clinical parameters as predictors of 90-days mortality in patients admitted with sepsis at Humanitas Research Hospital. Methods: Plasma samples were collected from 178 patients, diagnosed based on Sepsis-3 criteria, at admission to the Emergency Department and after 5 days of hospitalization. Levels of pentraxin 3 (PTX3), soluble IL-1 type 2 receptor (sIL-1R2), and of a panel of pro- and anti-inflammatory cytokines were measured by ELISA. Cox proportional-hazard models were used to evaluate predictors of 90-days mortality. Results: Circulating levels of PTX3, sIL-1R2, IL-1ß, IL-6, IL-8, IL-10, IL-18, IL-1ra, TNF-α increased significantly in sepsis patients on admission, with the highest levels measured in shock patients, and correlated with SOFA score (PTX3: r=0.44, p<0.0001; sIL-1R2: r=0.35, p<0.0001), as well as with 90-days mortality. After 5 days of hospitalization, PTX3 and cytokines, but not sIL-1R2 levels, decreased significantly, in parallel with a general improvement of clinical parameters. The combination of age, blood urea nitrogen, PTX3, IL-6 and IL-18, defined a prognostic index predicting 90-days mortality in Sepsis-3 patients and showing better apparent discrimination capacity than the SOFA score (AUC=0.863, 95% CI: 0.780-0.945 vs. AUC=0.727, 95% CI: 0.613-0.840; p=0.021 respectively). Conclusion: These data suggest that a prognostic index based on selected cytokines, PTX3 and clinical parameters, and hence easily adoptable in clinical practice, performs in predicting 90-days mortality better than SOFA. An independent validation is required.

Intratumoral combination therapy with poly(I:C) and resiquimod synergistically triggers tumor-associated macrophages for effective systemic antitumoral immunity.

BACKGROUND: Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of most treatments currently applied in the clinic. Previous studies have evaluated the antitumoral immune response triggered by (TLR) agonists, such as poly(I:C), imiquimod (R837) or resiquimod (R848) as monotherapies; however, their combination for the treatment of cancer has not been explored. This study investigates the antitumoral efficacy and the macrophage reprogramming triggered by poly(I:C) combined with R848 or with R837, versus single treatments. METHODS: TLR agonist treatments were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry using primary human and murine M-CSF-differentiated macrophages. Cytotoxic activity of TLR-treated macrophages toward cancer cells was evaluated with an in vitro functional assay by flow cytometry. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry, RT-qPCR, multispectral immunophenotyping, quantitative proteomic experiments, and protein-protein interaction analysis. RESULTS: Results demonstrated the higher efficacy of poly(I:C) combined with R848 versus single treatments or combined with R837 to polarize macrophages toward M1-like antitumor effectors in vitro. In vivo, the intratumoral synergistic combination of poly(I:C)+R848 significantly prevented tumor growth and metastasis in lung cancer and fibrosarcoma immunocompetent murine models. Regressing tumors showed increased infiltration of macrophages with a higher M1:M2 ratio, recruitment of CD4+ and CD8+ T cells, accompanied by a reduction of immunosuppressive CD206+ TAMs and FOXP3+/CD4+ T cells. The depletion of both CD4+ and CD8+ T cells resulted in complete loss of treatment efficacy. Treated mice acquired systemic antitumoral response and resistance to tumor rechallenge mediated by boosted macrophage cytotoxic activity and T-cell proliferation. Proteomic experiments validate the superior activation of innate immunity by poly(I:C)+R848 combination versus single treatments or poly(I:C)+R837, and protein-protein-interaction network analysis reveal the key activation of the STAT1 pathway. DISCUSSION: These findings demonstrate the antitumor immune responses mediated by macrophage activation on local administration of poly(I:C)+R848 combination and support the intratumoral application of this therapy to patients with solid tumors in the clinic.

Macrophage expression and prognostic significance of the long pentraxin PTX3 in COVID-19.

Long pentraxin 3 (PTX3) is an essential component of humoral innate immunity, involved in resistance to selected pathogens and in the regulation of inflammation1-3. The present study was designed to assess the presence and significance of PTX3 in Coronavirus Disease 2019 (COVID-19)4-7. RNA-sequencing analysis of peripheral blood mononuclear cells, single-cell bioinformatics analysis and immunohistochemistry of lung autopsy samples revealed that myelomonocytic cells and endothelial cells express high levels of PTX3 in patients with COVID-19. Increased plasma concentrations of PTX3 were detected in 96 patients with COVID-19. PTX3 emerged as a strong independent predictor of 28-d mortality in multivariable analysis, better than conventional markers of inflammation, in hospitalized patients with COVID-19. The prognostic significance of PTX3 abundance for mortality was confirmed in a second independent cohort (54 patients). Thus, circulating and lung myelomonocytic cells and endothelial cells are a major source of PTX3, and PTX3 plasma concentration can serve as an independent strong prognostic indicator of short-term mortality in COVID-19.

Complement C3 vs C5 inhibition in severe COVID-19: Early clinical findings reveal differential biological efficacy.

Growing clinical evidence has implicated complement as a pivotal driver of COVID-19 immunopathology. Deregulated complement activation may fuel cytokine-driven hyper-inflammation, thrombotic microangiopathy and NET-driven immunothrombosis, thereby leading to multi-organ failure. Complement therapeutics have gained traction as candidate drugs for countering the detrimental consequences of SARS-CoV-2 infection. Whether blockade of terminal complement effectors (C5, C5a, or C5aR1) may elicit similar outcomes to upstream intervention at the level of C3 remains debated. Here we compare the efficacy of the C5-targeting monoclonal antibody eculizumab with that of the compstatin-based C3-targeted drug candidate AMY-101 in small independent cohorts of severe COVID-19 patients. Our exploratory study indicates that therapeutic complement inhibition abrogates COVID-19 hyper-inflammation. Both C3 and C5 inhibitors elicit a robust anti-inflammatory response, reflected by a steep decline in C-reactive protein and IL-6 levels, marked lung function improvement, and resolution of SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). C3 inhibition afforded broader therapeutic control in COVID-19 patients by attenuating both C3a and sC5b-9 generation and preventing FB consumption. This broader inhibitory profile was associated with a more robust decline of neutrophil counts, attenuated neutrophil extracellular trap (NET) release, faster serum LDH decline, and more prominent lymphocyte recovery. These early clinical results offer important insights into the differential mechanistic basis and underlying biology of C3 and C5 inhibition in COVID-19 and point to a broader pathogenic involvement of C3-mediated pathways in thromboinflammation. They also support the evaluation of these complement-targeting agents as COVID-19 therapeutics in large prospective trials.

PLGA Based Nanoparticles for the Monocyte-Mediated Anti-Tumor Drug Delivery System.

Together with the development of new therapeutic agents, innovation in the delivery system of anti-tumor drugs is required to increase tumor-specificity and avoid unexpected toxicity. To achieve higher efficiency, we combined a live cell-mediated drug delivery system with nanotechnology, with the aim to prove that blood monocytes can be a cargo to deliver antitumor drugs encapsulated in Polymeric poly(D, L-lactide-co-glycolide) acid based nanoparticles (PLGA NPs). In this study, we have characterized how isolated purified monocytes efficiently internalize PLGA-NPs and have imaged in vivo their trafficking upon intravenous injection in tumor-bearing mice. Monocytes carrying PLGA-Cy7 NPs were able to reach the tumor site, with superior efficiency than free PLGA-Cy7 NPs, and the bio-distribution analysis confirmed that tumors were the most reached among peripheral tissues. We further demonstrate that monocytes carrying Doxorubicin encapsulated PLGA NPs (PLGA-Doxo) induced strong killing of co-cultured tumor cells. Our studies provide proof-of-concept evidence that monocytes can be exploited in approaches of live cell-mediated drug delivery systems for tumor therapy.

The macrophage tetraspan MS4A4A enhances dectin-1-dependent NK cell-mediated resistance to metastasis.

The plasma membrane tetraspan molecule MS4A4A is selectively expressed by macrophage-lineage cells, but its function is unknown. Here we report that MS4A4A was restricted to murine and human mononuclear phagocytes and was induced during monocyte-to-macrophage differentiation in the presence of interleukin 4 or dexamethasone. Human MS4A4A was co-expressed with M2/M2-like molecules in subsets of normal tissue-resident macrophages, infiltrating macrophages from inflamed synovium and tumor-associated macrophages. MS4A4A interacted and colocalized with the ß-glucan receptor dectin-1 in lipid rafts. In response to dectin-1 ligands, Ms4a4a-deficient macrophages showed defective signaling and defective production of effector molecules. In experimental models of tumor progression and metastasis, Ms4a4a deficiency in macrophages had no impact on primary tumor growth, but was essential for dectin-1-mediated activation of macrophages and natural killer (NK) cell-mediated metastasis control. Thus, MS4A4A is a tetraspan molecule selectively expressed in macrophages during differentiation and polarization, essential for dectin-1-dependent activation of NK cell-mediated resistance to metastasis.

Pentraxin 3 deficiency protects from the metabolic inflammation associated to diet-induced obesity.

AIMS: Low-grade chronic inflammation characterizes obesity and metabolic syndrome. Here, we aim at investigating the impact of the acute-phase protein long pentraxin 3 (PTX3) on the immune-inflammatory response occurring during diet-induced obesity. METHODS AND RESULTS: PTX3 deficiency in mice fed a high-fat diet for 20 weeks protects from weight gain and adipose tissue deposition in visceral and subcutaneous depots. This effect is not related to changes in glucose homeostasis and lipid metabolism but is associated with an improved immune cell phenotype in the adipose tissue of Ptx3 deficient animals, which is characterized by M2-macrophages polarization and increased angiogenesis. These findings are recapitulated in humans where carriers of a PTX3 haplotype (PTX3 h2/h2 haplotype), resulting in lower PTX3 plasma levels, presented with a reduced prevalence of obesity and decreased abdominal adiposity compared with non-carriers. CONCLUSION: Our results support a critical role for PTX3 in the onset of obesity by promoting inflammation and limiting adipose tissue vascularization and delineate PTX3 targeting as a valuable strategy for the treatment of adipose tissue-associated inflammatory response.

The Long Pentraxin 3 Plays a Role in Bone Turnover and Repair.

Pentraxin 3 (PTX3) is an inflammatory mediator acting as a fluid-phase pattern recognition molecule and playing an essential role in innate immunity and matrix remodeling. Inflammatory mediators also contribute to skeletal homeostasis, operating at multiple levels in physiological and pathological conditions. This study was designed to investigate the role of PTX3 in physiological skeletal remodeling and bone healing. Micro-computed tomography (µCT) and bone histomorphometry of distal femur showed that PTX3 gene-targeted female and male mice (ptx3-/- ) had lower trabecular bone volume than their wild-type (ptx3+/+ ) littermates (BV/TV by µCT: 3.50 ± 1.31 vs 6.09 ± 1.17 for females, p < 0.0001; BV/TV 9.06 ± 1.89 vs 10.47 ± 1.97 for males, p = 0.0435). In addition, µCT revealed lower trabecular bone volume in second lumbar vertebra of ptx3-/- mice. PTX3 was increasingly expressed during osteoblast maturation in vitro and was able to reverse the negative effect of fibroblast growth factor 2 (FGF2) on osteoblast differentiation. This effect was specific for the N-terminal domain of PTX3 that contains the FGF2-binding site. By using the closed transversal tibial fracture model, we found that ptx3-/- female mice formed significantly less mineralized callus during the anabolic phase following fracture injury compared to ptx3+/+ mice (BV/TV 17.05 ± 4.59 vs 20.47 ± 3.32, p = 0.0195). Non-hematopoietic periosteal cells highly upregulated PTX3 expression during the initial phase of fracture healing, particularly CD51+ and αSma+ osteoprogenitor subsets, and callus tissue exhibited concomitant expression of PTX3 and FGF2 around the fracture site. Thus, PTX3 supports maintenance of the bone mass possibly by inhibiting FGF2 and its negative impact on bone formation. Moreover, PTX3 enables timely occurring sequence of callus mineralization after bone fracture injury. These results indicate that PTX3 plays an important role in bone homeostasis and in proper matrix mineralization during fracture repair, a reflection of the function of this molecule in tissue homeostasis and repair.

ACKR2 in hematopoietic precursors as a checkpoint of neutrophil release and anti-metastatic activity.

Atypical chemokine receptors (ACKRs) are regulators of leukocyte traffic, inflammation, and immunity. ACKR2 is a scavenger for most inflammatory CC chemokines and is a negative regulator of inflammation. Here we report that ACKR2 is expressed in hematopoietic precursors and downregulated during myeloid differentiation. Genetic inactivation of ACKR2 results in increased levels of inflammatory chemokine receptors and release from the bone marrow of neutrophils with increased anti-metastatic activity. In a model of NeuT-driven primary mammary carcinogenesis ACKR2 deficiency is associated with increased primary tumor growth and protection against metastasis. ACKR2 deficiency results in neutrophil-mediated protection against metastasis in mice orthotopically transplanted with 4T1 mammary carcinoma and intravenously injected with B16F10 melanoma cell lines. Thus, ACKR2 is a key regulator (checkpoint) of mouse myeloid differentiation and function and its targeting unleashes the anti-metastatic activity of neutrophils in mice.

Pentraxin 3 plasma levels at graft-versus-host disease onset predict disease severity and response to therapy in children given haematopoietic stem cell transplantation.

Acute Graft-versus-Host Disease (GvHD) remains a major complication of allogeneic haematopoietic stem cell transplantation, with a significant proportion of patients failing to respond to first-line systemic corticosteroids. Reliable biomarkers predicting disease severity and response to treatment are warranted to improve its management. Thus, we sought to determine whether pentraxin 3 (PTX3), an acute-phase protein produced locally at the site of inflammation, could represent a novel acute GvHD biomarker. Using a murine model of the disease, we found increased PTX3 plasma levels after irradiation and at GvHD onset. Similarly, plasma PTX3 was enhanced in 115 pediatric patients on day of transplantation, likely due to conditioning, and at GvHD onset in patients experiencing clinical symptoms of the disease. PTX3 was also found increased in skin and colon biopsies from patients with active disease. Furthermore, PTX3 plasma levels at GvHD onset were predictive of disease outcome since they resulted significantly higher in both severe and therapy-unresponsive patients. Multiple injections of rhPTX3 in the murine model of GvHD did not influence the disease course. Taken together, our results indicate that PTX3 constitutes a biomarker of GvHD severity and therapy response useful to tailor treatment intensity according to early risk-stratification of GvHD patients.

Functional TRAIL receptors in monocytes and tumor-associated macrophages: A possible targeting pathway in the tumor microenvironment.

Despite the accepted dogma that TRAIL kills only tumor cells and spares normal ones, we show in this study that mononuclear phagocytes are susceptible to recombinant TRAIL via caspase-dependent apoptosis. Human resting monocytes and in vitro-differentiated macrophages expressed substantial levels of the functional TRAIL receptors (TRAIL-R1 and TRAIL-R2), while neutrophils and lymphocytes mostly expressed the non-signaling decoy receptor (TRAIL-R3). Accordingly, exclusively monocytes and macrophages activated caspase-8 and underwent apoptosis upon recombinant TRAIL treatment. TRAIL-Rs were up-regulated by anti-inflammatory agents (IL-10, glucocorticoids) and by natural compounds (Apigenin, Quercetin, Palmitate) and their treatment resulted in increased TRAIL-induced apoptosis. In mice, the only signaling TRAIL-R (DR5) was preferentially expressed by blood monocytes rather than neutrophils or lymphocytes. In both mice and humans, Tumor-Associated Macrophages (TAM) expressed functional TRAIL-R, while resident macrophages in normal tissues did not. As a proof of principle, we treated mice bearing a murine TRAIL-resistant fibrosarcoma with recombinant TRAIL. We observed significant decrease of circulating monocytes and infiltrating TAM, as well as reduced tumor growth and lower metastasis formation. Overall, these findings demonstrate that human and murine monocytes/macrophages are, among leukocytes, uniquely susceptible to TRAIL-mediated killing. This differential susceptibility to TRAIL could be exploited to selectively target macrophages in tumors.

An acidic microenvironment sets the humoral pattern recognition molecule PTX3 in a tissue repair mode.

Pentraxin 3 (PTX3) is a fluid-phase pattern recognition molecule and a key component of the humoral arm of innate immunity. In four different models of tissue damage in mice, PTX3 deficiency was associated with increased fibrin deposition and persistence, and thicker clots, followed by increased collagen deposition, when compared with controls. Ptx3-deficient macrophages showed defective pericellular fibrinolysis in vitro. PTX3-bound fibrinogen/fibrin and plasminogen at acidic pH and increased plasmin-mediated fibrinolysis. The second exon-encoded N-terminal domain of PTX3 recapitulated the activity of the intact molecule. Thus, a prototypic component of humoral innate immunity, PTX3, plays a nonredundant role in the orchestration of tissue repair and remodeling. Tissue acidification resulting from metabolic adaptation during tissue repair sets PTX3 in a tissue remodeling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity.

Endothelial cell-derived chemerin promotes dendritic cell transmigration.

ChemR23 is a chemotactic receptor expressed by APCs, such as dendritic cells, macrophages, and NK cells. Chemerin, the ChemR23 ligand, was detected by immunohistochemistry, to be associated with inflamed endothelial cells in autoimmune diseases, such as lupus erythematosus, psoriasis, and rheumatoid arthritis. This study reports that blood and lymphatic murine endothelial cells produce chemerin following retinoic acid stimulation. Conversely, proinflammatory cytokines, such as TNF-α, IFN-γ, and LPS, or calcitriol, are not effective. Retinoic acid-stimulated endothelial cells promoted dendritic cell adhesion under shear stress conditions and transmigration in a ChemR23-dependent manner. Activated endothelial cells upregulated the expression of the atypical chemotactic receptor CCRL2/ACKR5, a nonsignaling receptor able to bind and present chemerin to ChemR23(+) dendritic cells. Accordingly, activated endothelial cells expressed chemerin on the plasma membrane and promoted in a more efficient manner chemerin-dependent transmigration of dendritic cells. Finally, chemerin stimulation of myeloid dendritic cells induced the high-affinity binding of VCAM-1/CD106 Fc chimeric protein and promoted VCAM-1-dependent arrest to immobilized ligands under shear stress conditions. In conclusion, this study reports that retinoic acid-activated endothelial cells can promote myeloid and plasmacytoid dendritic cell transmigration across endothelial cell monolayers through the endogenous production of chemerin, the upregulation of CCRL2, and the activation of dendritic cell ß1 integrin affinity.

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.

Blocking TH17-polarizing cytokines by histone deacetylase inhibitors in vitro and in vivo.

Histone deacetylase (HDAC) inhibitors are small molecules inducing cell-cycle arrest, differentiation, and apoptosis, currently undergoing clinical trials as anticancer drugs. In addition, emerging evidence suggests HDAC inhibitors may have anti-inflammatory and immunomodulatory properties as well, although the molecular mechanisms remain poorly defined. Given the central role of dendritic cells (DC) in the induction and maintenance of the inflammatory and immune response, we investigated the effects of HDAC inhibitors on the maturation and activation of human monocyte-derived DC in the presence of LPS and IFN-gamma. Our results show that the production of T(H)1- and T(H)17-inducing cytokines, namely IL-12 and IL-23, was inhibited by trichostatin A (72% and 52%, respectively) and suberoylanilide hydroxamic acid (86% and 83%). Strikingly, HDAC inhibitors were effective if added simultaneously as well as after the proinflammatory challenge, and their effect was not associated to a reduction of expression or function of LPS/IFN-gamma receptors. These findings were confirmed in two different murine models. In addition, HDAC inhibitors selectively blocked the production of T(H)1-attracting chemokines CXCL9, CXCL10, and CXCL11. The reduction of T(H)1- and T(H)17-inducing cytokines as well as T(H)1-attracting chemokines may represent relevant mechanisms through which HDAC inhibitors at nonproapoptotic doses exert their immunomodulatory properties.

Cell-specific regulation of PTX3 by glucocorticoid hormones in hematopoietic and nonhematopoietic cells.

PTX3 (prototypic long pentraxin 3) is a fluid phase pattern recognition receptor, which plays nonredundant roles in the resistance against diverse pathogens, in the assembly of a hyaluronic acid-rich extracellular matrix, and in female fertility. Inflammatory signals induce production of PTX3 in diverse cell types, including myeloid dendritic cells (DC), fibroblasts, and endothelial cells (EC). The present study was designed to explore the effect of glucocorticoid hormones (GC) on PTX3 production in different cellular contexts. In myeloid DC, GC inhibited the PTX3 production. In contrast, in fibroblasts and EC, GC alone induced and, under inflammatory conditions, enhanced and extended PTX3 production. In vivo administration of GC augmented the blood levels of PTX3 in mice and humans. Moreover, patients with Cushing syndrome had increased levels of circulating PTX3, whereas PTX3 levels were decreased in subjects affected by iatrogenic hypocortisolism. In nonhematopoietic cells, GC receptor (GR) functioned as a ligand-dependent transcription factor (dimerization-dependent) to induce PTX3 gene expression. In contrast, in hematopoietic cells, GR repressed PTX3 gene transcription by interfering (dimerization-independent) with the action of other signaling pathways, probably NFkappaB and AP-1. Thus, divergent effects of GC were found to be due to different GR mechanisms. The results presented here indicate that GC have divergent effects on PTX3 production in hematopoietic (DC and macrophages) and nonhematopoietic (fibroblasts and EC) cells. The divergent effects of GC on PTX3 production probably reflect the different functions of this multifunctional molecule in innate immunity and in the construction of the extracellular matrix.