RESUMEN
Tenascin-C (TNC) is a complex glycoprotein of the extracellular matrix (ECM) involved in a plethora of (patho-)physiological processes, such as oncogenesis and inflammation. Since chemokines play an essential role in both disease processes, we have investigated here the binding of TNC to some of the key chemokines, namely CCL2, CCL26, CXCL8, CXCL10, and CXCL12. Thereby, a differential chemokine-TNC binding pattern was observed, with CCL26 exhibiting the highest and CCL2 the lowest affinity for TNC. Heparan sulfate (HS), another member of the ECM, proved to be a similarly high-affinity ligand of TNC, with a Kd value of 730 nM. Chemokines use glycosa-minoglycans such as HS as co-receptors to induce immune cell migration. Therefore, we assumed an influence of TNC on immune cell chemotaxis due to co-localization within the ECM. CCL26- and CCL2-induced mobilization experiments of eosinophils and monocytes, respectively, were thus performed in the presence and the absence of TNC. Pre-incubation of the immune cells with TNC resulted in a 3.5-fold increase of CCL26-induced eosinophil chemotaxis, whereas a 1.3-fold de-crease in chemotaxis was observed when monocytes were pre-incubated with CCL2. As both chemokines have similar HS binding but different TNC binding affinities, we speculate that TNC acts as an attenuator in monocyte and as an amplifier in eosinophil mobilization by impeding CCL2 from binding to HS on the one hand, and by reinforcing CCL26 to bind to HS on the other hand.
Asunto(s)
Matriz Extracelular , Tenascina , Movimiento Celular , Matriz Extracelular/metabolismo , Heparitina Sulfato/metabolismo , Monocitos/metabolismo , Transducción de Señal , Tenascina/metabolismo , HumanosRESUMEN
Bidirectional dialogue between cellular and non-cellular components of the tumor microenvironment (TME) drives cancer survival. In the extracellular space, combinations of matrix molecules and soluble mediators provide external cues that dictate the behavior of TME resident cells. Often studied in isolation, integrated cues from complex tissue microenvironments likely function more cohesively. Here, we study the interplay between the matrix molecule tenascin-C (TNC) and chemokine CCL2, both elevated in and associated with the progression of breast cancer and playing key roles in myeloid immune responses. We uncover a correlation between TNC/CCL2 tissue levels in HER2+ breast cancer and examine the physical and functional interactions of these molecules in a murine disease model with tunable TNC levels and in in vitro cellular and cell-free models. TNC supported sustained CCL2 synthesis, with chemokine binding to TNC via two distinct domains. TNC dominated the behavior of tumor-resident myeloid cells; CCL2 did not impact macrophage survival/activation whilst TNC facilitated an immune suppressive macrophage phenotype that was not dependent on or altered by CCL2 co-expression. Together, these data map new binding partners within the TME and demonstrate that whilst the matrix exerts transcriptional control over the chemokine, each plays a distinct role in subverting anti-tumoral immunity.
Asunto(s)
Neoplasias , Tenascina , Animales , Ratones , Quimiocinas/metabolismo , Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Transducción de Señal , Tenascina/metabolismo , Quimiocina CCL2/metabolismoRESUMEN
Eruptive epidermoid cysts are a rare adverse event of imiquimod treatment for basal cell carcinoma. Up to date, 8 cases have been described in the literature. We present the case of a 75-year-old Caucasian woman with recurrent basal cell carcinoma on the nose. After multiple excisions and treatment with vismodegib, imiquimod 5% cream was administered 5 times per week over 6 weeks. Two months after the end of treatment, the patient presented with eruptive epidermoid cysts.
Asunto(s)
Antineoplásicos , Carcinoma Basocelular , Quiste Epidérmico , Imiquimod , Neoplasias Cutáneas , Anciano , Aminoquinolinas , Antineoplásicos/efectos adversos , Carcinoma Basocelular/tratamiento farmacológico , Quiste Epidérmico/inducido químicamente , Femenino , Humanos , Imiquimod/efectos adversos , Neoplasias Cutáneas/tratamiento farmacológico , Resultado del TratamientoRESUMEN
The pro-inflammatory chemokine interleukin-8 (CXCL8) exerts its function by establishing a chemotactic gradient in infected or damaged tissues to guide neutrophil granulocytes to the site of inflammation via its G protein-coupled receptors (GPCRs) CXCR1 and CXCR2 located on neutrophils. Endothelial glycosaminoglycans (GAGs) have been proposed to support the chemotactic gradient formation and thus the inflammatory response by presenting the chemokine to approaching leukocytes. In this study, we show that neutrophil transmigration in vitro can be reduced by adding soluble GAGs and that this process is specific with respect to the nature of the glycan. To further investigate the GAG influence on neutrophil migration, we have used an engineered CXCL8 mutant protein (termed PA401) which exhibits a much higher affinity towards GAGs and an impaired GPCR activity. This dominant-negative mutant chemokine showed anti-inflammatory activity in various animal models of neutrophil-driven inflammation, i.e. in urinary tract infection, bleomycin-induced lung fibrosis, and experimental autoimmune uveitis. In all cases, treatment with PA401 resulted in a strong reduction of transmigrated inflammatory cells which became evident from histology sections and bronchoalveolar lavage. Since our CXCL8-based decoy targets GAGs and not GPCRs, our results show for the first time the crucial involvement of this glycan class in CXCL8/neutrophil-mediated inflammation and will thus pave the way to novel approaches of anti-inflammatory treatment.
Asunto(s)
Glicosaminoglicanos/inmunología , Mediadores de Inflamación/inmunología , Neutrófilos/inmunología , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Interleucina-8/inmunología , Interleucina-8/farmacología , Neutrófilos/patología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Migración Transendotelial y Transepitelial/inmunologíaRESUMEN
Chemokine binding to glycosaminoglycans (GAGs) is recognised to be an important step in inflammation and other pathological disorders like tumor growth and metastasis. Although different ways and strategies to interfere with these interactions are being pursued, no major breakthrough in the development of glycan-targeting drugs has been reported so far. We have engineered CXCL8 towards a dominant-negative form of this chemokine (dnCXCL8) which was shown to be highly active in various inflammatory animal models due to its inability to bind/activate the cognate CXCL8 GPC receptors on neutrophils in combination with its significantly increased GAG-binding affinity [1]. For the development of GAG-targeting chemokine-based biopharmaceuticals, we have established a repertoire of methods which allow the quantification of protein-GAG interactions. Isothermal fluorescence titration (IFT), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and a novel ELISA-like competition assay (ELICO) have been used to determine Kd and IC50 values for CXCL8 and dnCXCL8 interacting with heparin and heparan sulfate (HS), the proto-typical members of the GAG family. Although the different methods gave different absolute affinities for the four protein-ligand pairs, the relative increase in GAG-binding affinity of dnCXCL8 compared to the wild type chemokine was found by all methods. In combination, these biophysical methods allow to discriminate between unspecific and specific protein-GAG interactions.
Asunto(s)
Antiinflamatorios/farmacología , Diseño de Fármacos , Glicosaminoglicanos/farmacología , Interleucina-8/farmacología , Receptores CXCR/metabolismo , Animales , Línea Celular , Glicosaminoglicanos/genética , Humanos , Inflamación/tratamiento farmacológico , Interleucina-8/genética , Unión Proteica , Ingeniería de ProteínasRESUMEN
BACKGROUND: Heart failure (HF) remains one of the leading causes of death to date despite extensive research funding. Various studies are conducted every year in an attempt to improve diagnostic accuracy and therapy monitoring. The small cytoplasmic heart-type fatty acid-binding protein (H-FABP) has been studied in a variety of disease entities. Here, we provide a review of the available literature on H-FABP and its possible applications in HF. Methods: Literature research using PubMed Central was conducted. To select possible studies for inclusion, the authors screened all available studies by title and, if suitable, by abstract. Relevant manuscripts were read in full text. RESULTS: In total, 23 studies regarding H-FABP in HF were included in this review. CONCLUSION: While, algorithms already exist in the area of risk stratification for acute pulmonary embolism, there is still no consensus for the routine use of H-FABP in daily clinical practice in HF. At present, the strongest evidence exists for risk evaluation of adverse cardiac events. Other future applications of H-FABP may include early detection of ischemia, worsening of renal failure, and long-term treatment planning.
RESUMEN
The interplay between cancer cells and immune cells is a key determinant of tumor survival. Here, we uncovered how tumors exploit the immunomodulatory properties of the extracellular matrix to create a microenvironment that enables their escape from immune surveillance. Using orthotopic grafting of mammary tumor cells in immunocompetent mice and autochthonous models of breast cancer, we discovered how tenascin-C, a matrix molecule absent from most healthy adult tissues but expressed at high levels and associated with poor patient prognosis in many solid cancers, controls the immune status of the tumor microenvironment. We found that, although host-derived tenascin-C promoted immunity via recruitment of proinflammatory, antitumoral macrophages, tumor-derived tenascin-C subverted host defense by polarizing tumor-associated macrophages toward a pathogenic, immune-suppressive phenotype. Therapeutic monoclonal antibodies that blocked tenascin-C activation of Toll-like receptor 4 reversed this phenotypic switch in vitro and reduced tumor growth and lung metastasis in vivo, providing enhanced benefit in combination with anti-PD-L1 over either treatment alone. Combined tenascin-C:macrophage gene-expression signatures delineated a significant survival benefit in people with breast cancer. These data revealed a new approach to targeting tumor-specific macrophage polarization that may be effective in controlling the growth and spread of breast tumors.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Macrófagos/inmunología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/inmunología , Femenino , Humanos , Vigilancia Inmunológica , Inmunoterapia/métodos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Ratones , Fenotipo , Tenascina/inmunología , Células Tumorales Cultivadas , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
The mechanisms by which protein complexes convert from functional to pathogenic are the subject of intensive research. Here, we report how functionally unfavorable protein interactions can be induced by structural fuzziness, i.e., by persisting conformational disorder in protein complexes. We show that extreme disorder in the bound state transforms the intrinsically disordered protein SERF1a from an RNA-organizing factor into a pathogenic enhancer of alpha-synuclein (aSyn) amyloid toxicity. We demonstrate that SERF1a promotes the incorporation of RNA into nucleoli and liquid-like artificial RNA-organelles by retaining an unusually high degree of conformational disorder in the RNA-bound state. However, this type of structural fuzziness also determines an undifferentiated interaction with aSyn. RNA and aSyn both bind to one identical, positively charged site of SERF1a by an analogous electrostatic binding mode, with similar binding affinities, and without any observable disorder-to-order transition. The absence of primary or secondary structure discriminants results in SERF1a being unable to select between nucleic acid and amyloidogenic protein, leading the pro-amyloid aSyn:SERF1a interaction to prevail in the cytosol under conditions of cellular stress. We suggest that fuzzy disorder in SERF1a complexes accounts for an adverse gain-of-interaction which favors toxic binding to aSyn at the expense of nontoxic RNA binding, thereby leading to a functionally distorted and pathogenic process. Thus, structural fuzziness constitutes a direct link between extreme conformational flexibility, amyloid aggregation, and the malfunctioning of RNA-associated cellular processes, three signatures of neurodegenerative proteinopathies.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , ARN/química , alfa-Sinucleína/metabolismo , Animales , Citosol/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Ratones , Proteínas del Tejido Nervioso/química , Ácidos Nucleicos/química , Unión Proteica , ARN/metabolismo , Electricidad Estática , alfa-Sinucleína/químicaRESUMEN
Monocyte chemoattractant protein-1 (MCP-1/CCL2) is renowned for its ability to drive the chemotaxis of myeloid and lymphoid cells. It orchestrates the migration of these cell types both during physiological immune defense and in pathological circumstances, such as autoimmune diseases including rheumatoid arthritis and multiple sclerosis, inflammatory diseases including atherosclerosis, as well as infectious diseases, obesity, diabetes, and various types of cancer. However, new data suggest that the scope of CCL2's functions may extend beyond its original characterization as a chemoattractant. Emerging evidence shows that it can impact leukocyte behavior, influencing adhesion, polarization, effector molecule secretion, autophagy, killing, and survival. The direction of these CCL2-induced responses is context dependent and, in some cases, synergistic with other inflammatory stimuli. The involvement of CCL2 signaling in multiple diseases renders it an interesting therapeutic target, although current targeting strategies have not met early expectations in the clinic. A better understanding of how CCL2 affects immune cells will be pivotal to the improvement of existing therapeutic approaches and the development of new drugs. Here, we provide an overview of the pleiotropic effects of CCL2 signaling on cells of the myeloid lineage, beyond chemotaxis, and highlight how these actions might help to shape immune cell behavior and tumor immunity.
Asunto(s)
Quimiocina CCL2/metabolismo , Quimiotaxis/inmunología , Células Mieloides/inmunología , Células Mieloides/metabolismo , Animales , Comunicación Celular , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Quimiocina CCL2/genética , Quimiotaxis/genética , Regulación de la Expresión Génica , Humanos , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Transducción de SeñalRESUMEN
The chemokine CXCL10 is released by a plethora of cells, including immune and metastatic cancer cells, following stimulation with interferon-gamma. It acts via its GPC receptor on T-cells attracting them to various target tissues. Glycosaminoglycans (GAGs) are regarded as co-receptors of chemokines, which enable the establishment of a chemotactic gradient for target cell migration. We have engineered human CXCL10 towards improved T-cell mobilisation by implementing a single site-directed mutation N20K into the protein, which leads to a higher GAG binding affinity compared to the wild type. Interestingly, this mutation not only increased T-cell migration in a transendothelial migration assay, the mutant intensified T-cell chemotaxis also in a Boyden chamber set-up thereby indicating a strong role of T-cell-localised GAGs on leukocyte migration. A CXCL10 mutant with increased GAG-binding affinity could therefore potentially serve as a T-cell mobiliser in pathological conditions where the immune surveillance of the target tissue is impaired, as is the case for most solid tumors.
Asunto(s)
Quimiocina CXCL10/metabolismo , Glicosaminoglicanos/metabolismo , Simulación de Dinámica Molecular , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Movimiento Celular , Células Cultivadas , Quimiocina CXCL10/química , Quimiocina CXCL10/genética , Quimiotaxis de Leucocito , Glicosaminoglicanos/química , Humanos , Masculino , Mutación Missense , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas/métodosRESUMEN
Multiple Sclerosis, a chronic inflammatory demyelinating disease of the central nervous system, involves an increased expression of monocyte chemotactic protein 1 MCP1-/CCL2. For exerting its chemotactic effects, chemokine binding to glycosaminoglycans (GAGs) is required and therefore this interaction represents a potential target for therapeutic intervention. We have designed an anti-inflammatory decoy variant, Met-CCL2 (Y13A S21K Q23R), embodying increased affinity for GAGs as well as knocked-out GPCR activation properties. This non-signalling dominant-negative mutant is shown here to be able to displace wild type CCL2 from GAGs by which it is supposed to interfere with the chemokine-related inflammatory response. In vivo, the anti-inflammatory properties were successfully demonstrated in a murine model of zymosan-induced peritonitis as well as in an experimental autoimmune encephalomyelitis, a model relevant for multiple sclerosis, where the compound lead to significantly reduced clinical scores due to reduction of cellular infiltrates and demyelination in spinal cord and cerebellum. These findings indicate a promising potential for future therapeutic development.
Asunto(s)
Antiinflamatorios/administración & dosificación , Quimiocina CCL2/administración & dosificación , Encefalitis/prevención & control , Glicosaminoglicanos/química , Animales , Antiinflamatorios/farmacocinética , Cerebelo/efectos de los fármacos , Cerebelo/patología , Quimiocina CCL2/genética , Quimiocina CCL2/farmacocinética , Dexametasona/administración & dosificación , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/prevención & control , Femenino , Concentración 50 Inhibidora , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos , Peritonitis/inducido químicamente , Peritonitis/prevención & control , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , ZimosanRESUMEN
Interactions between chemokines and glycosaminoglycans (GAGs) are crucial for the physiological and pathophysiological activities of chemokines. GAGs are therefore commonly designated as chemokine coreceptors which are deeply involved in the chemokine-signaling network. Studying the interaction of chemokines with GAGs is therefore a major prerequisite to fully understand the biological function of chemokines. GAGs are, however, a very complex class of biomacromolecules which cannot be produced by conventional recombinant methods and which, if purchased from commercial suppliers, are often not subjected to rigorous quality control and therefore frequently differ in batch characteristics. This naturally impacts chemokine-GAG interaction studies. In order to standardize the quality of our GAG ligands, we have therefore established protocols for the preparation and characterization of GAGs from various cells and tissues, for which we give practical examples relating to the major GAG classes heparin, heparan sulfate, and chondroitin sulfate. We will also outline robust and sensitive protocols for chemokine-GAG interaction studies. By this means, a better and more common understanding of the involvement of GAGs in chemokine-signaling networks can be envisaged.
Asunto(s)
Glicosaminoglicanos/aislamiento & purificación , Glicosaminoglicanos/metabolismo , Biología Molecular/métodos , Receptores de Quimiocina/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Fluorescencia , Heparitina Sulfato/química , Heparitina Sulfato/aislamiento & purificación , Heparitina Sulfato/metabolismo , Humanos , MamíferosRESUMEN
We have engineered GPCR (G protein-coupled receptor) knock-out and high GAG-binding affinity into CXCL12α to inhibit CXCL12α-induced cell migration. Compared to wtCXCL12, the mutant CXCL12α (Δ8 L29K V39K) exhibited a 5.6-fold and a 2.2-fold affinity increase for heparin and heparan sulfate, respectively. From NaCl-based heparin displacement chromatography we concluded that more amino acid replacements would lead to altered GAG (glycosaminoglycan) ligand specificity. GAG silencing by this mutant was shown in a murine seeding model of human cancer cells, whereby a greatly reduced number of liver metastases was detected when the animals were treated intravenously with 1mg/kg CXCL12α (Δ8 L29K V39K) before cancer cell application.