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OBJECTIVE: To use cell-based gene signatures to identify patients with systemic lupus erythematous (SLE) in the phase II/III APRIL-SLE and phase IIb ADDRESS II trials most likely to respond to atacicept. METHODS: A published immune cell deconvolution algorithm based on Affymetrix gene array data was applied to whole blood gene expression from patients entering APRIL-SLE. Five distinct patient clusters were identified. Patient characteristics, biomarkers, and clinical response to atacicept were assessed per cluster. A modified immune cell deconvolution algorithm was developed based on RNA sequencing data and applied to ADDRESS II data to identify similar patient clusters and their responses. RESULTS: Patients in APRIL-SLE (N = 105) were segregated into the following five clusters (P1-5) characterized by dominant cell subset signatures: high neutrophils, T helper cells and natural killer (NK) cells (P1), high plasma cells and activated NK cells (P2), high B cells and neutrophils (P3), high B cells and low neutrophils (P4), or high activated dendritic cells, activated NK cells, and neutrophils (P5). Placebo- and atacicept-treated patients in clusters P2,4,5 had markedly higher British Isles Lupus Assessment Group (BILAG) A/B flare rates than those in clusters P1,3, with a greater treatment effect of atacicept on lowering flares in clusters P2,4,5. In ADDRESS II, placebo-treated patients from P2,4,5 were less likely to be SLE Responder Index (SRI)-4, SRI-6, and BILAG-Based Combined Lupus Assessment responders than those in P1,3; the response proportions again suggested lower placebo effect and a greater treatment differential for atacicept in P2,4,5. CONCLUSION: This exploratory analysis indicates larger differences between placebo- and atacicept-treated patients with SLE in a molecularly defined patient subset.
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Toll-like receptor (TLR) 7 and TLR8 are transmembrane receptors that recognize single-stranded RNA. Activation of these receptors results in immune cell stimulation and inflammatory cytokine production, which is normally a protective host response. However, aberrant activation of TLR7/8 is potentially pathogenic and linked to progression of certain autoimmune diseases such as lupus. Thus, we hypothesize that an inhibitor that blocks TLR7/8 would be an effective therapeutic treatment. Prior efforts to develop inhibitors of TLR7/8 have been largely unsuccessful as a result of the challenge of producing a small-molecule inhibitor for these difficult targets. Here, we report the characterization of M5049 and compound 2, molecules which were discovered in a medicinal chemistry campaign to produce dual TLR7/8 inhibitors with drug-like properties. Both compounds showed potent and selective activity in a range of cellular assays for inhibition of TLR7/8 and block synthetic ligands and natural endogenous RNA ligands such as microRNA and Alu RNA. M5049 was found to be potent in vivo as TLR7/8 inhibition efficaciously treated disease in several murine lupus models and, interestingly, was efficacious in a disease context in which TLR7/8 activity has not previously been considered a primary disease driver. Furthermore, M5049 had greater potency in disease models than expected based on its in vitro potency and pharmacokinetic/pharmacodynamic properties. Because of its preferential accumulation in tissues, and ability to block multiple TLR7/8 RNA ligands, M5049 may be efficacious in treating autoimmunity and has the potential to provide benefit to a variety of patients with varying disease pathogenesis. SIGNIFICANCE STATEMENT: This study reports discovery of a novel toll-like receptor (TLR) 7 and TLR8 inhibitor (M5049); characterizes its binding mode, potency/selectivity, and pharmacokinetic and pharmacodynamic properties; and demonstrates its potential for treating autoimmune diseases in two mouse lupus models. TLR7/8 inhibition is unique in that it may block both innate and adaptive autoimmunity; thus, this study suggests that M5049 has the potential to benefit patients with autoimmune diseases.
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Autoinmunidad/efectos de los fármacos , Descubrimiento de Drogas , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 8/antagonistas & inhibidores , Animales , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Conformación Proteica , Receptor Toll-Like 7/química , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/química , Receptor Toll-Like 8/metabolismoRESUMEN
Anti-CD20-mediated B-cell depletion effectively reduces acute multiple sclerosis (MS) flares. Recent data shows that antibody-mediated extinction of B cells as a lasting immune suppression, harbors the risk of developing humoral deficiencies over time. Accordingly, more selective, durable and reversible B-cell-directed MS therapies are needed. We here tested inhibition of Bruton's tyrosine kinase (BTK), an enzyme centrally involved in B-cell receptor signaling, as the most promising approach in this direction. Using mouse models of MS, we determined that evobrutinib, the first BTK inhibiting molecule being developed, dose-dependently inhibited antigen-triggered activation and maturation of B cells as well as their release of pro-inflammatory cytokines. Most importantly, evobrutinib treatment functionally impaired the capacity of B cells to act as antigen-presenting cells for the development of encephalitogenic T cells, resulting in a significantly reduced disease severity in mice. In contrast to anti-CD20, BTK inhibition silenced this key property of B cells in MS without impairing their frequency or functional integrity. In conjunction with a recent phase II trial reporting that evobrutinib is safe and effective in MS, our mechanistic data highlight therapeutic BTK inhibition as a landmark towards selectively interfering with MS-driving B-cell properties.
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Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Linfocitos B/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/inmunología , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/enzimología , Encefalomielitis Autoinmune Experimental/patología , Humanos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Because of its role in mediating both B cell and Fc receptor signaling, Bruton's tyrosine kinase (BTK) is a promising target for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Evobrutinib is a novel, highly selective, irreversible BTK inhibitor that potently inhibits BCR- and Fc receptor-mediated signaling and, thus, subsequent activation and function of human B cells and innate immune cells such as monocytes and basophils. We evaluated evobrutinib in preclinical models of RA and SLE and characterized the relationship between BTK occupancy and inhibition of disease activity. In mouse models of RA and SLE, orally administered evobrutinib displayed robust efficacy, as demonstrated by reduction of disease severity and histological damage. In the SLE model, evobrutinib inhibited B cell activation, reduced autoantibody production and plasma cell numbers, and normalized B and T cell subsets. In the RA model, efficacy was achieved despite failure to reduce autoantibodies. Pharmacokinetic/pharmacodynamic modeling showed that mean BTK occupancy in blood cells of 80% was linked to near-complete disease inhibition in both RA and SLE mouse models. In addition, evobrutinib inhibited mast cell activation in a passive cutaneous anaphylaxis model. Thus, evobrutinib achieves efficacy by acting both on B cells and innate immune cells. Taken together, our data show that evobrutinib is a promising molecule for the chronic treatment of B cell-driven autoimmune disorders.
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Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Artritis Reumatoide/tratamiento farmacológico , Linfocitos B/inmunología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Activación de Linfocitos/efectos de los fármacos , Piperidinas/farmacología , Pirimidinas/farmacología , Agammaglobulinemia Tirosina Quinasa/inmunología , Animales , Artritis Reumatoide/enzimología , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Linfocitos B/enzimología , Linfocitos B/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Lupus Eritematoso Sistémico/enzimología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Ratones , Células U937RESUMEN
BACKGROUND: Pharmacodynamic biomarkers are becoming increasingly valuable for assessing drug activity and target modulation in clinical trials. However, identifying quality biomarkers is challenging due to the increasing volume and heterogeneity of relevant data describing the biological networks that underlie disease mechanisms. A biological pathway network typically includes entities (e.g. genes, proteins and chemicals/drugs) as well as the relationships between these and is typically curated or mined from structured databases and textual co-occurrence data. We propose a hybrid Natural Language Processing and directed relationships-based network analysis approach using IBM Watson for Drug Discovery to rank all human genes and identify potential candidate biomarkers, requiring only an initial determination of a specific target-disease relationship. METHODS: Through natural language processing of scientific literature, Watson for Drug Discovery creates a network of semantic relationships between biological concepts such as genes, drugs, and diseases. Using Bruton's tyrosine kinase as a case study, Watson for Drug Discovery's automatically extracted relationship network was compared with a prominent manually curated physical interaction network. Additionally, potential biomarkers for Bruton's tyrosine kinase inhibition were predicted using a matrix factorization approach and subsequently compared with expert-generated biomarkers. RESULTS: Watson's natural language processing generated a relationship network matching 55 (86%) genes upstream of BTK and 98 (95%) genes downstream of Bruton's tyrosine kinase in a prominent manually curated physical interaction network. Matrix factorization analysis predicted 11 of 13 genes identified by Merck subject matter experts in the top 20% of Watson for Drug Discovery's 13,595 ranked genes, with 7 in the top 5%. CONCLUSION: Taken together, these results suggest that Watson for Drug Discovery's automatic relationship network identifies the majority of upstream and downstream genes in biological pathway networks and can be used to help with the identification and prioritization of pharmacodynamic biomarker evaluation, accelerating the early phases of disease hypothesis generation.
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Biomarcadores/análisis , Descubrimiento de Drogas/métodos , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Agammaglobulinemia Tirosina Quinasa/genética , Agammaglobulinemia Tirosina Quinasa/metabolismo , Área Bajo la Curva , Bases de Datos Factuales , Humanos , Redes y Vías Metabólicas , Procesamiento de Lenguaje Natural , Curva ROC , Bibliotecas de Moléculas Pequeñas/farmacocinéticaRESUMEN
The TNF family cytokines B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) support plasma cell survival. It is known that inhibitors of BAFF only (BAFFR-Fc) or BAFF and APRIL (TACI-Fc) administered early enough in an NZB/NZW F1 mouse model of systemic lupus erythematosus (SLE) ameliorate clinical outcomes, pointing to a pathogenic role of BAFF. In the present study, TACI-Fc administrated at a later stage of disease, after onset of autoimmunity, decreased the number of bone marrow plasma cells and slowed down further formation of autoantibodies. TACI-Fc prevented renal damage during a 12-week treatment period regardless of autoantibody levels, while BAFFR-Fc did not despite a similar BAFF-blocking activity in vivo. TACI-Fc also decreased established plasma cells in a T-dependent hapten/carrier immunization system better than single inhibitors of BAFF or APRIL, and sometimes better than combined single inhibitors with at least equivalent BAFF and APRIL inhibitory activities. These results indicate that TACI-Fc can prevent symptoms of renal damage in a mouse model of SLE when BAFFR-Fc cannot, and point to a plasticity of plasma cells for survival factors. Targeting plasma cells with TACI-Fc might be beneficial to prevent autoantibody-mediated damages in SLE.
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Modelos Animales de Enfermedad , Lupus Eritematoso Sistémico/inmunología , Células Plasmáticas/inmunología , Proteína Activadora Transmembrana y Interactiva del CAML/administración & dosificación , Animales , Autoanticuerpos/biosíntesis , Autoinmunidad , Factor Activador de Células B/antagonistas & inhibidores , Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/administración & dosificación , Receptor del Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Citometría de Flujo , Riñón/inmunología , Riñón/patología , Lupus Eritematoso Sistémico/fisiopatología , Lupus Eritematoso Sistémico/terapia , Ratones , Células Plasmáticas/patología , Proteína Activadora Transmembrana y Interactiva del CAML/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/antagonistas & inhibidores , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunologíaRESUMEN
Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large number of Btk inhibitors have been developed. These compounds have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a molecular level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compounds can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcεR signaling as Y551 sequestering compounds were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in determining potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compounds with a broader inhibitory profile that will likely result in greater efficacy.
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Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores Fc/metabolismo , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismo , Agammaglobulinemia Tirosina Quinasa , Línea Celular Tumoral , Análisis por Conglomerados , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismoRESUMEN
SLE is a complex autoimmune inflammatory disease characterized by pathogenic autoantibody production as a consequence of uncontrolled T-B cell activity and immune-complex deposition in various organs, including kidney, leading to tissue damage and function loss. There is a high unmet need for better treatment options other than corticosteroids and immunosuppressants. Phosphoinositol-3 kinase δ (PI3Kδ) is a promising target in this respect as it is essential in mediating B- and T-cell function in mouse and human. We report the identification of selective PI3Kδ inhibitors that blocked B-, T-, and plasmacytoid dendritic cell activities in human peripheral blood and in primary cell co-cultures (BioMAP(®)) without detecting signs of undesired toxicity. In an IFNα-accelerated mouse SLE model, our PI3Kδ inhibitors blocked nephritis development, whether administered at the onset of autoantibody appearance or the onset of proteinuria. Disease amelioration correlated with normalized immune cell numbers in the spleen, reduced immune-complex deposition as well as reduced inflammation, fibrosis, and tissue damage in the kidney. Improvements were similar to those achieved with a frequently prescribed drug for lupus nephritis, the potent immunosuppressant mycophenolate mofetil. Finally, we established a pharmacodynamics/pharmacokinetic/efficacy model that revealed that a sustained PI3Kδ inhibition of 50% is sufficient to achieve full efficacy in our disease model. These data demonstrate the therapeutic potential of PI3Kδ inhibitors in SLE and lupus nephritis.
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The anti-phospholipid syndrome (APS) is characterized by recurrent thrombosis and occurrence of anti-phospholipid antibodies (aPL). aPL are necessary, but not sufficient for the clinical manifestations of APS. Growing evidence suggests a role of innate immune cells, in particular polymorphonuclear neutrophils (PMN) and Toll-like receptors (TLR) to be additionally involved. aPL activate endothelial cells and monocytes through a TLR4-dependent signalling pathway. Whether this is also relevant for PMN in a similar way is currently not known. To address this issue, we used purified PMN from healthy donors and stimulated them in the presence or absence of human monoclonal aPL and the TLR4 agonist LPS monitoring neutrophil effector functions, namely the oxidative burst, phagocytosis, L-Selectin shedding and IL-8 production. aPL alone were only able to induce minor activation of PMN effector functions at high concentrations. However, in the additional presence of LPS the activation threshold was markedly lower indicating a synergistic activation pathway of aPL and TLR in PMN. In summary, our results indicate that PMN effector functions are directly activated by aPL and boosted by the additional presence of microbial products. This highlights a role for PMN as important innate immune effector cells that contribute to the pathophysiology of APS.
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Síndrome Antifosfolípido/fisiopatología , Neutrófilos/metabolismo , Transducción de Señal/fisiología , Receptor Toll-Like 4/fisiología , Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/metabolismo , Apoptosis , Antígeno CD11b/inmunología , Citometría de Flujo , Humanos , Interleucina-8/biosíntesis , Selectina L/metabolismo , Lipopolisacáridos/farmacología , Activación Neutrófila , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Fagocitosis , Estallido RespiratorioRESUMEN
The triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in the innate inflammatory response to microbial infections. Activation and expression of TREM-1 by polymorphonuclear neutrophils (PMN) occurs in concert with Toll-like receptors (TLR) such as TLR4 for bacterial lipopolysaccharide. However, it is currently unclear how this is mediated on a molecular level. Using pharmacological inhibitors and Western blot analysis we demonstrate that phosphatidyl inositide 3-kinase, phospholipase C and the mitogen-activated kinase p38MAPK are essential for the TREM-1- and TLR4-induced oxidative burst of human PMN. The activation of protein kinase B and extracellular signal-related kinase show characteristic phosphorylation patterns upon single or co-ligation indicating individual activation pathways of both receptors. Taken together, we provide new insights into the mechanisms how TREM-1 and TLR interact creating synergistic activation in PMN. These results shed a new light on our understanding of how the innate inflammatory responses are regulated and might contribute to the development of future concepts for the treatment of severe inflammatory conditions such as sepsis.
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Glicoproteínas de Membrana/metabolismo , Neutrófilos/inmunología , Receptores Inmunológicos/metabolismo , Estallido Respiratorio/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/metabolismo , Western Blotting , Separación Celular , Activación Enzimática/inmunología , Citometría de Flujo , Humanos , Glicoproteínas de Membrana/inmunología , Modelos Moleculares , Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Inmunológicos/inmunología , Receptor Toll-Like 4/inmunología , Receptor Activador Expresado en Células Mieloides 1 , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The triggering receptor expressed on myeloid cells 1 (TREM-1) plays an important role in the innate immune response related to severe infections and sepsis. Modulation of TREM-1-associated activation improves the outcome in rodent models for pneumonia and sepsis. However, the identity and occurrence of the natural TREM-1 ligands are so far unknown, impairing the further understanding of the biology of this receptor. Here, we report the presence of a ligand for TREM-1 on human platelets. Using a recombinant TREM-1 fusion protein, we demonstrate specific binding of TREM-1 to platelets. TREM-1-specific signals are required for the platelet-induced augmentation of polymorphonuclear leukocyte (PMN) effector functions (provoked by LPS). However, TREM-1 interaction with its ligand is not required for platelet/PMN complex formation, which is dependent on integrins and selectins. Taken together, the results indicate that the TREM-1 ligand is expressed by platelets, and the TREM-1/ligand interaction contributes to the amplification of LPS-induced PMN activation. Our results shed new light on our understanding of TREM-1 and its role in the innate inflammatory response in infections and might contribute to the development of future concepts to treat sepsis.
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Plaquetas/metabolismo , Ligandos , Glicoproteínas de Membrana/farmacología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunidad Innata/efectos de los fármacos , Integrinas/metabolismo , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Neumonía/tratamiento farmacológico , Neumonía/metabolismo , Unión Proteica/efectos de los fármacos , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Selectinas/metabolismo , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Receptor Activador Expresado en Células Mieloides 1RESUMEN
Chronic obstructive pulmonary disease (COPD) is increasingly recognized as a systemic disease that is associated with increased serum levels of markers of systemic inflammation. The triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently identified activating receptor on neutrophils, monocytes, and macrophage subsets. TREM-1 expression is upregulated by microbial products such as the toll-like receptor ligand lipoteichoic acid of Gram-positive or lipopolysaccharides of Gram-negative bacteria. In the present study, sera from 12 COPD patients (GOLD stages I-IV, FEV1 51 +/- 6%) and 10 healthy individuals were retrospectively analyzed for soluble TREM-1 (sTREM-1) using a newly developed ELISA. In healthy subjects, sTREM-1 levels were low (median 0.25 ng/mL, range 0-5.9 ng/mL). In contrast, levels of sTREM-1 in sera of COPD patients were significantly increased (median 11.68 ng/mL, range 6.2-41.9 ng/mL, P<.05). Furthermore, serum levels of sTREM-1 showed a significant negative correlation with lung function impairment. In summary, serum concentrations of sTREM-1 are increased in patients with COPD. Prospective studies are warranted to evaluate the relevance of sTREM-1 as a potential marker of the disease in patients with COPD.
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Biomarcadores/sangre , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Receptores Inmunológicos/sangre , Ensayo de Inmunoadsorción Enzimática , Humanos , Enfermedad Pulmonar Obstructiva Crónica/patología , Proteínas Recombinantes/sangre , Proteínas Recombinantes/metabolismo , Pruebas de Función Respiratoria , Estudios Retrospectivos , Transfección , Receptor Activador Expresado en Células Mieloides 1RESUMEN
In microbial infections polymorphnuclear neutrophils (PMN) constitute a major part of the innate host defence, based upon their ability to rapidly accumulate in inflamed tissues and clear the site of infection from microbial pathogens by their potent effector mechanisms. The recently described transmembrane receptor herpes virus entry mediator (HVEM) is a member of the tumour necrosis factor receptor super family and is expressed on many haematopoietic cells, including T cells, B cells, natural killer cells, monocytes and PMN. Interaction of HVEM with the natural ligand LIGHT on T cells has a costimulatory effect, and increases the bactericidal activity of PMN. To further characterize the function of HVEM on PMN, we evaluated the effect of receptor ligation on human PMN effector functions using an agonistic monoclonal antibody. Here we demonstrate that activation of HVEM causes activation of neutrophil effector functions, including respiratory burst, degranulation and release of interleukin-8 in synergy with ligands for Toll-like receptors or GM-CSF. In addition, stimulation via HVEM enhanced neutrophil phagocytic activity of complement opsonized, but not of non-opsonized, particles. In conclusion, these results indicate a new, as yet unknown, participation of HVEM in the innate immune response and points to a new link between innate and adaptive immunity.
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Activación Neutrófila/inmunología , Neutrófilos/inmunología , Miembro 14 de Receptores del Factor de Necrosis Tumoral/inmunología , Receptores Toll-Like/inmunología , Degranulación de la Célula/inmunología , Supervivencia Celular/inmunología , Células Cultivadas , Humanos , Mediadores de Inflamación/inmunología , Interleucina-8/metabolismo , Ligandos , Fagocitosis/inmunología , Estallido Respiratorio/inmunologíaRESUMEN
Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free (<0.5 enzyme units/mg). As described previously, innate immune cells are activated by Gp96 at high concentrations (> or =50 microg/ml) but not at lower concentrations. However, preincubation of low amounts of Gp96 with TLR2 and TLR4 ligands at concentrations unable to activate dendritic cells by themselves results in the production of high levels of proinflammatory cytokines, up-regulation of activation markers, and amplification of T cell activation. Our results provide significant new insights into the mechanism of HSP-mediated dendritic cell activation and present a new function of HSPs in the amplification of dendritic cell activation by bacterial products and induction of adaptive immune responses.