RESUMEN
The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.
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COVID-19/inmunología , Inmunidad Humoral , Receptores de Reconocimiento de Patrones/inmunología , SARS-CoV-2/inmunología , Animales , Proteína C-Reactiva/inmunología , Proteína C-Reactiva/metabolismo , COVID-19/metabolismo , COVID-19/virología , Estudios de Casos y Controles , Chlorocebus aethiops , Activación de Complemento , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Femenino , Glicosilación , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Masculino , Lectina de Unión a Manosa/genética , Lectina de Unión a Manosa/inmunología , Lectina de Unión a Manosa/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Polimorfismo Genético , Unión Proteica , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Componente Amiloide P Sérico/inmunología , Componente Amiloide P Sérico/metabolismo , Transducción de Señal , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células VeroRESUMEN
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.
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Proteína C-Reactiva/genética , COVID-19/genética , Perfilación de la Expresión Génica/métodos , Macrófagos/metabolismo , SARS-CoV-2/aislamiento & purificación , Componente Amiloide P Sérico/genética , Células A549 , Adulto , Proteína C-Reactiva/metabolismo , COVID-19/epidemiología , COVID-19/virología , Línea Celular Tumoral , Células Cultivadas , Estudios de Cohortes , Células Endoteliales/metabolismo , Epidemias , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Neutrófilos/metabolismo , Pronóstico , SARS-CoV-2/fisiología , Componente Amiloide P Sérico/metabolismoRESUMEN
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.
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Células Asesinas Naturales/inmunología , Lectinas Tipo C/metabolismo , Macrófagos/inmunología , Proteínas de la Membrana/metabolismo , Metástasis de la Neoplasia/inmunología , Neoplasias/inmunología , Animales , Diferenciación Celular/inmunología , Linaje de la Célula , Dexametasona/farmacología , Humanos , Interleucina-4/metabolismo , Activación de Linfocitos/inmunología , Activación de Macrófagos/inmunología , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Metástasis de la Neoplasia/prevención & control , Neoplasias/patologíaRESUMEN
Pentraxins are a superfamily of conserved proteins involved in the acute-phase response and innate immunity. Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a key component of the humoral arm of innate immunity that is essential for resistance to certain pathogens. A regulatory role for pentraxins in inflammation has long been recognized, but the underlying mechanisms remain unclear. Here we report that PTX3 bound P-selectin and attenuated neutrophil recruitment at sites of inflammation. PTX3 released from activated leukocytes functioned locally to dampen neutrophil recruitment and regulate inflammation. Antibodies have glycosylation-dependent regulatory effect on inflammation. Therefore, PTX3, which is an essential component of humoral innate immunity, and immunoglobulins share functional outputs, including complement activation, opsonization and, as shown here, glycosylation-dependent regulation of inflammation.
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Proteína C-Reactiva/inmunología , Inflamación/inmunología , Rodamiento de Leucocito/inmunología , Infiltración Neutrófila/inmunología , Componente Amiloide P Sérico/inmunología , Lesión Pulmonar Aguda/inmunología , Animales , Células CHO , Separación Celular , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Humanos , Inmunidad Humoral/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Recombinantes/inmunologíaRESUMEN
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.
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Betacoronavirus/patogenicidad , Complemento C3/antagonistas & inhibidores , Complemento C5/antagonistas & inhibidores , Inactivadores del Complemento/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Neumonía Viral/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Biomarcadores/sangre , Proteína C-Reactiva/metabolismo , COVID-19 , Estudios de Cohortes , Activación de Complemento/efectos de los fármacos , Complemento C3/genética , Complemento C3/inmunología , Complemento C5/genética , Complemento C5/inmunología , Infecciones por Coronavirus/complicaciones , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Trampas Extracelulares/efectos de los fármacos , Femenino , Expresión Génica , Humanos , Interleucina-6/metabolismo , Masculino , Persona de Mediana Edad , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/virología , Pandemias , Péptidos Cíclicos/uso terapéutico , Neumonía Viral/complicaciones , Neumonía Viral/inmunología , Neumonía Viral/virología , Síndrome de Dificultad Respiratoria/complicaciones , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/virología , SARS-CoV-2 , Índice de Severidad de la EnfermedadRESUMEN
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.
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Factores Quimiotácticos/inmunología , Células Dendríticas/inmunología , Células Endoteliales/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Migración Transendotelial y Transepitelial/inmunología , Animales , Antineoplásicos/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Adhesión Celular/inmunología , Línea Celular , Quimiocinas , Factores Quimiotácticos/genética , Células Dendríticas/citología , Células Endoteliales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Noqueados , Receptores CCR , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/inmunología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Migración Transendotelial y Transepitelial/genética , Tretinoina/farmacología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
INTRODUCTION: Timely diagnosis of pneumonia in intubated critically ill patients is rather challenging. Pentraxin 3 (PTX3) is an acute-phase mediator produced by various cell types in the lungs. Animal studies have shown that, during pneumonia, PTX3 participates in fine-tuning of inflammation (for example, microbial clearance and recruitment of neutrophils). We previously described an association between alveolar PTX3 and lung infection in a small group of intubated patients. The aim of the present study was to determine a threshold level of alveolar PTX3 with elevated sensitivity and specificity for microbiologically confirmed pneumonia. METHODS: We recruited 82 intubated patients from two intensive care units (San Gerardo Hospital, Monza, Italy, and Massachusetts General Hospital, Boston, MA, USA) undergoing bronchoalveolar lavage (BAL) as per clinical decision. We collected BAL fluid and plasma samples, together with relevant clinical and microbiological data. We assayed PTX3 and soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) in BAL fluid and PTX3, sTREM-1, C-reactive protein (CRP) and procalcitonin (PCT) in plasma. Two blinded independent physicians reviewed patient data to confirm pneumonia. We determined the PTX3 threshold in BAL fluid for pneumonia and compared it to other biomarkers. RESULTS: Microbiologically confirmed pneumonia of bacterial (n =12), viral (n =4) or fungal (n =8) etiology was diagnosed in 24 patients (29%). PTX3 levels in BAL fluid predicted pneumonia with an area under the receiving operator curve of 0.815 (95% CI =0.710 to 0.921, P <0.0001), whereas none of the other biomarkers were effective. In particular, PTX3 levels ≥1 ng/ml in BAL fluid predicted pneumonia in univariate analysis (ß =2.784, SE =0.792, P <0.001) with elevated sensitivity (92%), specificity (60%) and negative predictive value (95%). Net reclassification index PTX3 values ≥1 ng/ml in BAL fluid for pneumonia indicated gain in sensitivity and/or specificity vs. all other mediators. These results did not change when we limited our analyses only to confirmed cases of bacterial pneumonia. Moreover, when we considered only the 70 patients who fulfilled the clinical criteria for the diagnosis of pneumonia at BAL fluid sampling, the diagnostic accuracy of PTX levels was confirmed in univariate and ROC curve analysis. CONCLUSIONS: In this hypothesis-generating convenience sample, a PTX3 level ≥1 ng/ml in BAL fluid was discriminative of microbiologically confirmed pneumonia in mechanically ventilated patients.
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Líquido del Lavado Bronquioalveolar/citología , Proteína C-Reactiva/análisis , Neumonía Bacteriana/diagnóstico , Componente Amiloide P Sérico/análisis , Adulto , Anciano , Biomarcadores/sangre , Líquido del Lavado Bronquioalveolar/microbiología , Calcitonina/sangre , Péptido Relacionado con Gen de Calcitonina , Diagnóstico Diferencial , Femenino , Humanos , Unidades de Cuidados Intensivos , Masculino , Persona de Mediana Edad , Neumonía Bacteriana/sangre , Neumonía Bacteriana/microbiología , Neumonía Viral/diagnóstico , Estudios Prospectivos , Precursores de Proteínas/sangre , Curva ROC , Respiración Artificial , Sensibilidad y EspecificidadRESUMEN
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).
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Biomarcadores , Proteína C-Reactiva , COVID-19 , Coinfección , SARS-CoV-2 , Componente Amiloide P Sérico , Humanos , COVID-19/sangre , COVID-19/diagnóstico , Proteína C-Reactiva/metabolismo , Proteína C-Reactiva/análisis , Componente Amiloide P Sérico/metabolismo , Biomarcadores/sangre , Masculino , Femenino , Anciano , Persona de Mediana Edad , SARS-CoV-2/aislamiento & purificación , Infecciones Bacterianas/sangre , Infecciones Bacterianas/diagnóstico , Polipéptido alfa Relacionado con Calcitonina/sangre , Pronóstico , Micosis/sangre , Micosis/diagnóstico , Anciano de 80 o más AñosRESUMEN
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.
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Proteína C-Reactiva/metabolismo , Moléculas de Adhesión Celular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Neovascularización Fisiológica , Proteínas del Tejido Nervioso/metabolismo , Componente Amiloide P Sérico/metabolismo , Animales , Unión Competitiva , Proteína C-Reactiva/deficiencia , Proteína C-Reactiva/genética , Células CHO , Bovinos , Moléculas de Adhesión Celular/genética , Embrión de Pollo , Cricetinae , Cricetulus , Femenino , Factor 2 de Crecimiento de Fibroblastos/genética , Células HEK293 , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/metabolismo , Componente Amiloide P Sérico/genética , Resonancia por Plasmón de Superficie , TransfecciónRESUMEN
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.
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Inflamación , Infecciones Neumocócicas , Animales , Ratones , Inflamación/metabolismo , Neutrófilos/metabolismo , Fagocitosis , Infecciones Neumocócicas/genética , Infecciones Neumocócicas/metabolismo , Streptococcus pneumoniaeRESUMEN
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.
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Imidazoles , Neoplasias Pulmonares , Nanocápsulas , Humanos , Animales , Ratones , Macrófagos Asociados a Tumores , Manosa , Neoplasias Pulmonares/tratamiento farmacológico , Modelos Animales de Enfermedad , Protaminas , Microambiente TumoralRESUMEN
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.
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Interleucina-10 , Sepsis , Biomarcadores , Proteína C-Reactiva , Citocinas , Humanos , Recién Nacido , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1 , Interleucina-18 , Interleucina-6 , Interleucina-8 , Pronóstico , Curva ROC , Componente Amiloide P Sérico , Factor de Necrosis Tumoral alfaRESUMEN
The MS4A gene family encodes 18 tetraspanin-like proteins, most of which with unknown function. MS4A1 (CD20), MS4A2 (FcεRIß), MS4A3 (HTm4), and MS4A4A play important roles in immunity, whereas expression and function of other members of the family are unknown. The present investigation was designed to obtain an expression fingerprint of MS4A family members, using bioinformatics analysis of public databases, RT-PCR, and protein analysis when possible. MS4A3, MS4A4A, MS4A4E, MS4A6A, MS4A7, and MS4A14 were expressed by myeloid cells. MS4A6A and MS4A14 were expressed in circulating monocytes and decreased during monocyte-to-MÏ differentiation in parallel with an increase in MS4A4A expression. Analysis of gene expression regulation revealed a strong induction of MS4A4A, MS4A6A, MS4A7, and MS4A4E by glucocorticoid hormones. Consistently with in vitro findings, MS4A4A and MS4A7 were expressed in tissue MÏs from COVID-19 and rheumatoid arthritis patients. Interestingly, MS4A3, selectively expressed in myeloid precursors, was found to be a marker of immature circulating neutrophils, a cellular population associated to COVID-19 severe disease. The results reported here show that members of the MS4A family are differentially expressed and regulated during myelomonocytic differentiation, and call for assessment of their functional role and value as therapeutic targets.
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COVID-19 , Proteínas de la Membrana , Antígenos CD20 , Familia , Humanos , Proteínas de la Membrana/genética , Monocitos/metabolismoRESUMEN
Chemerin is a chemotactic agent that was recently identified as the ligand of ChemR23, a serpentine receptor expressed by activated macrophages and monocyte-derived dendritic cells (DCs). This paper shows that blood plasmacytoid and myeloid DCs express functional ChemR23. Recombinant chemerin induced the transmigration of plasmacytoid and myeloid DCs across an endothelial cell monolayer. In secondary lymphoid organs (lymph nodes and tonsils), ChemR23 is expressed by CD123(+) plasmacytoid DCs and by CD1a(+) DC-SIGN(+) DCs in the interfollicular T cell area. ChemR23(+) DCs were also observed in dermis from normal skin, whereas Langerhans cells were negative. Chemerin expression was selectively detected on the luminal side of high endothelial venules in secondary lymphoid organs and in dermal endothelial vessels of lupus erythematosus skin lesions. Chemerin(+) endothelial cells were surrounded by ChemR23(+) plasmacytoid DCs. Thus, ChemR23 is expressed and functional in plasmacytoid DCs, a property shared only by CXCR4 among chemotactic receptors. This finding, together with the selective expression of the cognate ligand on the luminal side of high endothelial venules and inflamed endothelium, suggests a key role of the ChemR23/chemerin axis in directing plasmacytoid DC trafficking.
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Células Dendríticas/fisiología , Lupus Eritematoso Sistémico/inmunología , Tejido Linfoide/irrigación sanguínea , Receptores de Quimiocina/fisiología , Piel/irrigación sanguínea , Movimiento Celular , Células Cultivadas , Quimiocinas/biosíntesis , Quimiocinas/farmacología , Factores Quimiotácticos/biosíntesis , Factores Quimiotácticos/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Endoteliales/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ligandos , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/patología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Células Mieloides/inmunología , Células Plasmáticas/inmunología , Receptores de Quimiocina/biosíntesis , Piel/inmunología , Piel/metabolismo , Piel/patología , Vénulas/inmunología , Vénulas/metabolismoRESUMEN
Innate immunity represents the first line of defence against pathogens and plays key roles in activation and orientation of the adaptive immune response. The innate immune system comprises both a cellular and a humoral arm. Components of the humoral arm include soluble pattern recognition molecules (PRMs) that recognise pathogen-associated molecular patterns (PAMPs) and initiate the immune response in coordination with the cellular arm, therefore acting as functional ancestors of antibodies. The long pentraxin PTX3 is a prototypic soluble PRM that is produced at sites of infection and inflammation by both somatic and immune cells. Gene targeting of this evolutionarily conserved protein has revealed a nonredundant role in resistance to selected pathogens. Moreover, PTX3 exerts important functions at the cross-road between innate immunity, inflammation, and female fertility. Here, we review the studies on PTX3, with emphasis on pathogen recognition and cross-talk with other components of the innate immune system.
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Proteína C-Reactiva/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Humoral/inmunología , Infecciones/inmunología , Inflamación/inmunología , Componente Amiloide P Sérico/inmunología , Proteína C-Reactiva/genética , Femenino , Fertilidad/genética , Fertilidad/inmunología , Humanos , Inmunidad Humoral/genética , Inmunidad Innata , Infecciones/genética , Inflamación/genética , Componente Amiloide P Sérico/genéticaRESUMEN
Long pentraxin PTX3, a pattern recognition molecule involved in innate immune responses, is upregulated by pro-inflammatory stimuli, contributors to secondary damage in traumatic brain injury (TBI). We analyzed PTX3 involvement in mice subjected to controlled cortical impact, a clinically relevant TBI mouse model. We measured PTX3 mRNA and protein in the brain and its circulating levels at different time point post-injury, and assessed behavioral deficits and brain damage progression in PTX3 KO mice. PTX3 circulating levels significantly increased 1-3 weeks after injury. In the brain, PTX3 mRNA was upregulated in different brain areas starting from 24 h and up to 5 weeks post-injury. PTX3 protein significantly increased in the brain cortex up to 3 weeks post-injury. Immunohistochemical analysis showed that, 48 h after TBI, PTX3 was localized in proximity of neutrophils, likely on neutrophils extracellular traps (NETs), while 1- and 2- weeks post-injury PTX3 co-localized with fibrin deposits. Genetic depletion of PTX3 did not affect sensorimotor deficits up to 5 weeks post-injury. At this time-point lesion volume and neuronal count, axonal damage, collagen deposition, astrogliosis, microglia activation and phagocytosis were not different in KO compared to WT mice. Members of the long pentraxin family, neuronal pentraxin 1 (nPTX1) and pentraxin 4 (PTX4) were also over-expressed in the traumatized brain, but not neuronal pentraxin 2 (nPTX2) or short pentraxins C-reactive protein (CRP) and serum amyloid P-component (SAP). The long-lasting pattern of activation of PTX3 in brain and blood supports its specific involvement in TBI. The lack of a clear-cut phenotype in PTX3 KO mice may depend on the different roles of this protein, possibly involved in inflammation early after injury and in repair processes later on, suggesting distinct functions in acute phases versus sub-acute or chronic phases. Brain long pentraxins, such as PTX4-shown here to be overexpressed in the brain after TBI-may compensate for PTX3 absence.
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Lesiones Encefálicas/metabolismo , Encéfalo/metabolismo , Proteína C-Reactiva/metabolismo , Neuronas/metabolismo , Componente Amiloide P Sérico/metabolismo , Regulación hacia Arriba , Animales , Lesiones Encefálicas/genética , Lesiones Encefálicas/patología , Proteína C-Reactiva/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Neuronas/patología , Neutrófilos/metabolismo , Componente Amiloide P Sérico/genéticaRESUMEN
Aims: To determine the relationship between PTX3 systemic and synovial levels and the clinical features of rheumatoid arthritis (RA) in a cohort of early, treatment naïve patients and to explore the relevance of PTX3 expression in predicting response to conventional-synthetic (cs) Disease-Modifying-Anti-Rheumatic-Drugs (DMARDs) treatment. Methods: PTX3 expression was analyzed in 119 baseline serum samples from early naïve RA patients, 95 paired samples obtained 6-months following the initiation of cs-DMARDs treatment and 43 healthy donors. RNA-sequencing analysis and immunohistochemistry for PTX3 were performed on a subpopulation of 79 and 58 synovial samples, respectively, to assess PTX3 gene and protein expression. Immunofluorescence staining was performed to characterize PTX3 expressing cells within the synovium. Results: Circulating levels of PTX3 were significantly higher in early RA compared to healthy donors and correlated with disease activity at baseline and with the degree of structural damages at 12-months. Six-months after commencing cs-DMARDs, a high level of PTX3, proportional to the baseline value, was still detectable in the serum of patients, regardless of their response status. RNA-seq analysis confirmed that synovial transcript levels of PTX3 correlated with disease activity and the presence of mediators of inflammation, tissue remodeling and bone destruction at baseline. PTX3 expression in the synovium was strongly linked to the degree of immune cell infiltration, the presence of ectopic lymphoid structures and seropositivity for autoantibodies. Accordingly, PTX3 was found to be expressed by numerous synovial cell types such as plasma cells, fibroblasts, vascular and lymphatic endothelial cells, macrophages, and neutrophils. The percentage of PTX3-positive synovial cells, although significantly reduced at 6-months post-treatment as a result of global decreased cellularity, was similar in cs-DMARDs responders and non-responders. Conclusion: This study demonstrates that, early in the disease and prior to treatment modification, the level of circulating PTX3 is a reliable marker of RA activity and predicts a high degree of structural damages at 12-months. In the joint, PTX3 associates with immune cell infiltration and the presence of ectopic lymphoid structures. High synovial and peripheral blood levels of PTX3 are associated with chronic inflammation characteristic of RA. Additional studies to determine the mechanistic link are required.
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Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Proteína C-Reactiva/análisis , Componente Amiloide P Sérico/análisis , Adulto , Anciano , Autoanticuerpos/sangre , Biomarcadores/metabolismo , Estudios de Casos y Controles , Femenino , Humanos , Inflamación/metabolismo , Masculino , Persona de Mediana Edad , Líquido Sinovial/metabolismo , Sinoviocitos/metabolismoRESUMEN
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.