RESUMEN
BACKGROUND: Patient-derived organoid (PDO) models offer potential to transform drug discovery for inflammatory bowel disease (IBD) but are limited by inconsistencies with differentiation and functional characterization. We profiled molecular and cellular features across a range of intestinal organoid models and examined differentiation and establishment of a functional epithelial barrier. METHODS: Patient-derived organoids or monolayers were generated from control or IBD patient-derived colon or ileum and were molecularly or functionally profiled. Biological or technical replicates were examined for transcriptional responses under conditions of expansion or differentiation. Cell-type composition was determined by deconvolution of cell-associated gene signatures and histological features. Differentiated control or IBD-derived monolayers were examined for establishment of transepithelial electrical resistance (TEER), loss of barrier integrity in response to a cocktail of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and prevention of cytokine-induced barrier disruption by the JAK inhibitor, tofacitinib. RESULTS: In response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut. Upon differentiation, both colon- and ileum-derived monolayers formed functional barriers, with sustained TEER. Barrier integrity was compromised by inflammatory cytokines IFN-γ and TNF-α, and damage was inhibited in a dose-dependent manner by tofacitinib. CONCLUSIONS: We describe the generation and characterization of human colonic or ileal organoid models capable of functional differentiation to mature epithelial cell types. In monolayer culture, these cells formed a robust epithelial barrier with sustained TEER and responses to pharmacological modulation. Our findings demonstrate that control and IBD patient-derived organoids possess consistent transcriptional and functional profiles that can enable development of epithelial-targeted therapies.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Intestinos , Organoides , Humanos , Citocinas/metabolismo , Células Epiteliales/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Organoides/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Intestinos/fisiologíaRESUMEN
IL-33 is a multifunctional cytokine that mediates local inflammation upon tissue damage. IL-33 is known to act on multiple cell types including group 2 innate lymphoid cells (ILC2s), Th2 cells, and mast cells to drive production of Th2 cytokines including IL-5 and IL-13. IL-33 signaling activity through transmembrane ST2L can be inhibited by soluble ST2 (sST2), which acts as a decoy receptor. Previous findings suggested that modulation of IL-13 levels in mice lacking decoy IL-13Rα2, or mice lacking IL-13, impacted responsiveness to IL-33. In this study, we used Il13 -/- mice to investigate whether IL-13 regulates IL-33 activity by modulating the transmembrane and soluble forms of ST2. In Il13 -/- mice, the effects of IL-33 administration were exacerbated relative to wild type (WT). Il13 -/- mice administered IL-33 i.p. had heightened splenomegaly, more immune cells in the peritoneum including an expanded ST2L+ ILC2 population, increased eosinophilia in the spleen and peritoneum, and reduced sST2 in the circulation and peritoneum. In the spleen, lung, and liver of mice given IL-33, gene expression of both isoforms of ST2 was increased in Il13 -/- mice relative to WT. We confirmed fibroblasts to be an IL-13-responsive cell type that can regulate IL-33 activity through production of sST2. This study elucidates the important regulatory activity that IL-13 exerts on IL-33 through induction of IL-33 decoy receptor sST2 and through modulation of ST2L+ ILC2s.
Asunto(s)
Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Animales , Citocinas , Inmunidad Innata , Interleucina-13 , Linfocitos/metabolismo , RatonesRESUMEN
There continues to be a major need for more effective inflammatory bowel disease (IBD) therapies. IL-13Rα2 is a decoy receptor that binds the cytokine IL-13 with high affinity and diminishes its STAT6-mediated effector functions. Previously, we found that IL-13Rα2 was necessary for IBD in mice deficient in the anti-inflammatory cytokine IL-10. Here, we tested for the first time a therapeutic antibody specifically targeting IL-13Rα2. We also used the antibody and Il13ra2-/- mice to dissect the role of IL-13Rα2 in IBD pathogenesis and recovery. Il13ra2-/- mice were modestly protected from induction of dextran sodium sulfate (DSS)-induced colitis. Following a 7-day recovery period, Il13ra2-/- mice or wild-type mice administered the IL-13Rα2-neutralizing antibody had significantly improved colon health compared to control mice. Neutralizing IL-13Rα2 to increase IL-13 bioavailability promoted resolution of IBD even if neutralization occurred only during recovery. To link our observations in mice to a large human cohort, we conducted a phenome-wide association study of a more active variant of IL-13 (R130Q) that has reduced affinity for IL-13Rα2. Human subjects carrying R130Q reported a lower risk for Crohn's disease. Our findings endorse moving anti-IL-13Rα2 into preclinical drug development with the goal of accelerating recovery and maintaining remission in Crohn's disease patients.
Asunto(s)
Antiinflamatorios/farmacología , Anticuerpos Monoclonales/farmacología , Enfermedades Inflamatorias del Intestino/metabolismo , Subunidad alfa2 del Receptor de Interleucina-13/antagonistas & inhibidores , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Animales , Enfermedad de Crohn/etiología , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Eosinófilos/inmunología , Eosinófilos/metabolismo , Mutación con Ganancia de Función , Variación Genética , Humanos , Inmunidad , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/patología , Subunidad alfa2 del Receptor de Interleucina-13/genética , Ratones , Oportunidad RelativaRESUMEN
Progressive multifocal leukoencephalopathy (PML) is a rare but serious disease. Caused by the JC virus (JCV), it occurs in individuals with weakened immune systems and is a potential adverse reaction for certain immunomodulatory drugs. The PML Consortium was created to find better methods to predict, prevent, and treat PML. The Consortium brought together the pharmaceutical industry with academic, regulatory, and patient communities to advance research and dialogue on PML through a not-for-profit, collaborative approach involving a grant program, scientific workshops and conferences, and disease awareness efforts. Over nearly a decade, the Consortium contributed to the PML and JCV fields by advancing research, scientific exchange, and awareness of PML. In addition to advancing knowledge and helping to build cross-sector consensus on research priorities, the Consortium's grant program filled a funding gap and brought new investigators into PML and JCV research. Additionally, the Consortium's workshops and conferences created platforms for exchange that drove dialogue on knowledge gaps and future research directions. The Consortium also contributed to the scientific knowledge base with two literature reviews, one on PML treatment studies and a second on T cell deficiencies as a risk factor for PML and the brain as a site for conversion of harmless JCV into a pathogenic virus. Finally, the Consortium addressed a significant information gap with its disease awareness website for healthcare professionals, patients, and caregivers. Beyond its impact on the PML and JCV fields, the PML Consortium is important because it provides a precedent for how the pharmaceutical industry, academic researchers, patient organizations, and government can work together to address rare diseases, in particular rare adverse events. This kind of collaboration could be replicated to speed progress in addressing other rare diseases and adverse events, with significant potential benefits for the scientific, medical, and patient communities. FUNDING: PML Consortium (PML Consortium, Washington, DC).
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/prevención & control , Leucoencefalopatía Multifocal Progresiva , Organizaciones sin Fines de Lucro , Humanos , Colaboración Intersectorial , Virus JC , Leucoencefalopatía Multifocal Progresiva/terapia , Leucoencefalopatía Multifocal Progresiva/virología , Investigación , Participación de los InteresadosRESUMEN
Interleukin-4 (IL-4) and IL-13 are critical drivers of immune activation and inflammation in ulcerative colitis, asthma and other diseases. Because these cytokines may have redundant function, dual targeting holds promise for achieving greater efficacy. We have recently described a bifunctional therapeutic targeting IL-4 and IL-13 developed on a novel protein scaffold, generated by combining specific binding domains in an optimal configuration using appropriate linker regions. In the current study, the bifunctional IL-4/IL-13 antagonist was evaluated in the murine oxazolone-induced colitis model, which produces disease with features of ulcerative colitis. The bifunctional IL-4/IL-13 antagonist reduced body weight loss throughout the 7-day course of the model, and ameliorated the increased colon weight and decreased colon length that accompany disease. Colon tissue gene expression was modulated in accordance with the treatment effect. Concentrations of serum amyloid P were elevated in proportion to disease severity, making it an effective biomarker. Serum concentrations of the bifunctional IL-4/IL-13 antagonist were inversely proportional to disease severity, colon tissue expression of pro-inflammatory genes, and serum amyloid P concentration. Taken together, these results define a panel of biomarkers signifying engagement of the IL-4/IL-13 pathway, confirm the T helper type 2 nature of disease in this model, and demonstrate the effectiveness of dual cytokine blockade.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Colitis Ulcerosa/metabolismo , Interleucina-13/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/farmacología , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Subunidad alfa2 del Receptor de Interleucina-13/antagonistas & inhibidores , Ratones , Oxazolona/efectos adversos , Proteínas Recombinantes de Fusión/administración & dosificación , Proteína Amiloide A Sérica/metabolismo , Componente Amiloide P Sérico/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
IL-4 and IL-13 comprise promising targets for therapeutic interventions in asthma and other Th2-associated diseases, but agents targeting either IL-4 or IL-13 alone have shown limited efficacy in human clinical studies. Because these cytokines may involve redundant function, dual targeting holds promise for achieving greater efficacy. We describe a bifunctional therapeutic targeting IL-4 and IL-13, developed by a combination of specific binding domains. IL-4-targeted and IL-13-targeted single chain variable fragments were joined in an optimal configuration, using appropriate linker regions on a novel protein scaffold. The bifunctional IL-4/IL-13 antagonist displayed high affinity for both cytokines. It was a potent and efficient neutralizer of both murine IL-4 and murine IL-13 bioactivity in cytokine-responsive Ba/F3 cells, and exhibited a half-life of approximately 4.7 days in mice. In a murine model of ovalbumin-induced ear swelling, the bifunctional molecule blocked both the IL-4/IL-13-dependent early-phase response and the IL-4-dependent late-phase response. In the ovalbumin-induced lung inflammation model, the bifunctional IL-4/IL-13 antagonist reduced the IL-4-dependent rise in serum IgE titers, and reduced IL-13-dependent airway hyperresponsiveness, lung inflammation, mucin gene expression, and serum chitinase responses. Taken together, these findings demonstrate the effective dual blockade of IL-4 and IL-13 with a single agent, which resulted in the modulation of a more extensive range of endpoints than could be achieved by targeting either cytokine alone.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inmunoglobulina E/inmunología , Interleucina-13/antagonistas & inhibidores , Interleucina-4/antagonistas & inhibidores , Neumonía/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/inmunología , Sitios de Unión , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/inmunología , Células CHO , Cricetinae , Reactivos de Enlaces Cruzados/química , Oído/fisiopatología , Femenino , Semivida , Subunidad alfa2 del Receptor de Interleucina-13/inmunología , Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Ratones , Ratones Endogámicos C57BL , Conformación Molecular , Pruebas de Neutralización , Ovalbúmina/efectos adversos , Ovalbúmina/inmunología , Neumonía/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Anticuerpos de Cadena Única/metabolismoRESUMEN
Human studies using Abs to two different, nonoverlapping epitopes of IL-13 suggested that epitope specificity can have a clinically significant impact on clearance of IL-13. We propose that Ab modulation of IL-13 interaction with IL-13Rα2 underlies this effect. Two Abs were administered to healthy subjects and mild asthmatics in separate dose-ranging studies and allergen-challenge studies. IMA-638 allows IL-13 interaction with IL-13Rα1 or IL-13Rα2 but blocks recruitment of IL-4Rα to the IL-13/IL-13Rα1 complex, whereas IMA-026 competes with IL-13 interaction with IL-13Rα1 and IL-13Rα2. We found â¼10-fold higher circulating titer of captured IL-13 in subjects treated with IMA-026 compared with those administered IMA-638. To understand how this difference could be related to epitope, we asked whether either Ab affects IL-13 internalization through cell surface IL-13Rα2. Humans inducibly express cell surface IL-13Rα2 but lack the soluble form that regulates IL-13 responses in mice. Cells with high IL-13Rα2 expression rapidly and efficiently depleted extracellular IL-13, and this activity persisted in the presence of IMA-638 but not IMA-026. The potency and efficiency of this clearance pathway suggest that cell surface IL-13Rα2 acts as a scavenger for IL-13. These findings could have important implications for the design and characterization of IL-13 antagonists.
Asunto(s)
Subunidad alfa2 del Receptor de Interleucina-13/metabolismo , Interleucina-13/inmunología , Interleucina-13/metabolismo , Isoanticuerpos/fisiología , Receptores Depuradores/metabolismo , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Relación Dosis-Respuesta Inmunológica , Sistemas de Liberación de Medicamentos , Espacio Extracelular/inmunología , Espacio Extracelular/metabolismo , Células HT29 , Humanos , Interleucina-13/antagonistas & inhibidores , Subunidad alfa2 del Receptor de Interleucina-13/antagonistas & inhibidores , Subunidad alfa2 del Receptor de Interleucina-13/biosíntesis , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Receptores Depuradores/antagonistas & inhibidores , Receptores Depuradores/fisiologíaRESUMEN
RATIONALE: Extensive evidence in animal models supports a role for IL-13 in the pathobiology of asthma. IMA-638 and IMA-026 are fully humanized IgG(1) antibodies that bind to different epitopes and neutralize IL-13 bioactivity. OBJECTIVES: We hypothesized that anti-IL-13 treatment would inhibit allergen-induced late-phase asthmatic responses, airway hyperresponsiveness, and inflammation in subjects with asthma. METHODS: Fifty-six subjects with mild, atopic asthma were recruited for two double-blind, randomized, placebo-controlled, parallel group trials to compare IMA-638 and IMA-026 IL-13 antibody treatments with placebo treatment. Drug was administered on Days 1 and 8, and allergen challenges were performed on Days 14 and 35. The primary outcome variable was the late-phase area under the curve (AUC), and secondary outcome variables were the early- and late-phase maximum percent fall in FEV(1), early AUC, allergen-induced shift in airway hyperresponsiveness, and sputum eosinophils. MEASUREMENTS AND MAIN RESULTS: The treatment difference with IMA-638 on Day 14 was -19.1 FEV(1) × hour (95% confidence interval: -36.2, -1.9) for the allergen-induced early AUC and -23.8 FEV(1) × hour (95% confidence interval: -46.4, -1.2) for the late AUC (both P < 0.05), but this effect was lost by Day 35. Treatment with IMA-026 did not attenuate the asthmatic responses on Day 14 or Day 35. There was no effect of either antibody on allergen-induced airway hyperresponsiveness or sputum eosinophils. The frequency of adverse events after administration of the IL-13 antibodies was similar to placebo. CONCLUSIONS: IL-13 has a role in allergen-induced airway responses in humans. Further study is required to determine whether anti-IL-13 monoclonal antibodies will be beneficial clinically.
Asunto(s)
Anticuerpos Neutralizantes/uso terapéutico , Asma/tratamiento farmacológico , Interleucina-13/antagonistas & inhibidores , Adolescente , Adulto , Alérgenos/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Método Doble Ciego , Femenino , Volumen Espiratorio Forzado , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/uso terapéutico , Interleucina-13/sangre , Interleucina-13/inmunología , Masculino , Persona de Mediana Edad , Esputo/citología , Esputo/inmunología , Resultado del Tratamiento , Adulto JovenRESUMEN
IL-13 is a Th2 cytokine that has been shown to be an important mediator of airway inflammation contributing to asthma lesions. Given its proposed role in asthma, measurements of this cytokine in serum may provide insights into disease mechanisms, progression and pharmacodynamic effects of IL-13 targeted therapeutics. However, current commercially available ELISA immunoassays are frequently unable to detect baseline concentrations of IL-13 in serum from healthy individuals, which are below the limit of detection. Here we describe the use of the novel microparticle-based Erenna IL-13 human immunoassay (Singulex, Inc.), which utilizes proprietary antibodies and single molecule counting technology, to quantify IL-13 from 100 microL of serum from apparently healthy subjects and clinically defined symptomatic and asymptomatic asthma subjects. The lower limit of quantification of the Erenna assay was validated at 0.07 pg/mL and the assay detected baseline concentrations of IL-13 in 98% of serum samples tested. The calibration curve showed good precision over the entire linear range of 0.07-50 pg/mL, with inter-assay imprecision <10% CV except at the lowest concentration tested (<15%). The intra- and inter-assay imprecision of spiked serum samples containing three different IL-13 concentrations (2, 8, and 25 pg/mL) ranged from 2.2-2.4% and 6.1-6.8%, respectively. Using the Erenna IL-13 assay, we observe that serum IL-13 concentrations range from <0.07-1.02 pg/mL in apparently healthy subjects (N=60) with similar ranges in asymptomatic (0.07-0.66 pg/mL, N=26) and symptomatic (<0.07-1.26 pg/mL, N=96) asthma subjects. The Erenna immunoassay improved sensitivity by over two full logs compared to previous ELISA methods, while using smaller sample volumes. In addition, the Erenna assay reliably measured IL-13 in endogenous and spiked human serum samples that were not quantifiable using other methods. Taken together, these results show that this novel assay offers a significant improvement over previous methods for high-sensitive quantitative measurement of IL-13 in human serum samples obtained from both apparently healthy and asthmatic subjects, and can be used in future clinical studies to accurately measure concentrations of this cytokine prior to and following drug therapy in human serum.
Asunto(s)
Asma/sangre , Interleucina-13/sangre , Juego de Reactivos para Diagnóstico , Calibración , Ensayo de Inmunoadsorción Enzimática/métodos , Técnica del Anticuerpo Fluorescente Directa/métodos , Técnica del Anticuerpo Fluorescente Directa/normas , Humanos , Interleucina-13/análisis , Sensibilidad y EspecificidadRESUMEN
In asthma, mast cells infiltrate the airway smooth muscle cell layer and secrete proinflammatory and profibrotic agents that contribute to airway remodeling. To study the effects of mast cell activation on smooth muscle cell-dependent matrix contraction, we developed coculture systems of human airway smooth muscle cells (HASM) with primary human mast cells derived from circulating progenitors or with the HMC-1 human mast cell line. Activation of primary human mast cells by IgE receptor cross-linking or activation of HMC-1 cells with C5a stimulated contraction of HASM-embedded collagen gels. Contractile activity could be transferred with conditioned medium from activated mast cells, implicating involvement of soluble factors. Cytokines and proteases are among the agents released by activated mast cells that may promote a contractile response. Both IL-13 and IL-6 enhanced contraction in this model and the activity of IL-13 was ablated under conditions leading to expression of the inhibitory receptor IL-13Ralpha2 on HASM. In addition to cytokines, matrix metalloproteinases (MMPs), and serine proteases induced matrix contraction. Inhibitor studies suggested that, although IL-13 could contribute to contraction driven by mast cell activation, MMPs were critical mediators of the response. Both MMP-1 and MMP-2 were strongly expressed in this system. Serine proteases also contributed to contraction induced by mast cell-activating agents and IL-13, most likely by mediating the proteolytic activation of MMPs. Hypercontractility is a hallmark of smooth muscle cells in the asthmatic lung. Our findings define novel mechanisms whereby mast cells may modulate HASM-driven contractile responses.
Asunto(s)
Mastocitos/fisiología , Contracción Muscular , Miocitos del Músculo Liso/fisiología , Comunicación Paracrina , Sistema Respiratorio/citología , Técnicas de Cocultivo , Colágeno , Citocinas/fisiología , Matriz Extracelular/enzimología , Humanos , Interleucina-13/fisiología , Interleucina-6/fisiología , Metaloproteasas/fisiología , Músculo Liso , Serina Endopeptidasas/fisiologíaRESUMEN
Mast cell-fibroblast interactions may contribute to fibrosis in asthma and other disease states. Fibroblast contraction is known to be stimulated by coculture with the human mast cell line, HMC-1, or by mast cell-derived agents. Matrix metalloproteinases (MMPs) can also mediate contraction, but the MMP-dependence of mast cell-induced fibroblast contractility is not established, and the consequences of mast cell activation within the coculture system have not been fully explored. We demonstrate that activation of primary human mast cells (pHMC) with IgE receptor cross-linking, or activation of HMC-1 with C5a, enhanced contractility of human lung fibroblasts in a three-dimensional collagen lattice system. This enhanced contractility was inhibited by the pan-MMP antagonist, batimastat, and was transferrable in the conditioned medium of activated mast cells. Exogenously added MMPs promoted gel contraction by mediating the proteolytic activation of latent transforming growth factor-beta (TGF-beta). Consistent with this, fibroblast contraction induced by mast cell activation was enhanced by addition of excess latent TGF-beta to the cultures. Batimastat inhibited this response, suggesting that MMPs capable of activating latent TGF-beta were released following mast cell activation in coculture with fibroblasts. Collagen production was also stimulated by activated mast cells in an MMP-dependent manner. MMP-2 and MMP-3 content of the gels increased in the presence of activated mast cells, and inhibition of these enzymes blocked the contractile response. These findings demonstrate the MMP dependence of mast cell-induced fibroblast contraction and collagen production.
Asunto(s)
Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Mastocitos/fisiología , Metaloproteinasas de la Matriz/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de los fármacos , Humanos , Inmunoglobulina E/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/genética , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Inhibidores de Proteasas/farmacología , Tiofenos/farmacología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Interleukin-13 (IL-13) is a critical mediator of asthma pathology. On B cells, monocytes, epithelial cells, and smooth muscle cells, IL-13 acts through the IL-13Ralpha1/IL-4Ralpha complex to directly induce activation responses that contribute to atopic disease. In human populations, genetic polymorphisms in IL-13, its receptor components, or the essential signaling element STAT6, have all been associated with increased risk of atopy and asthma. Animal studies using IL-13 deficient mice, IL-13 transgenic animals, and IL-13 neutralization strategies have confirmed an essential role for this cytokine in driving major correlates of asthma pathology, including airway hyperresponsiveness (AHR), lung eosinophilia, mucus generation, and fibrosis. Ongoing studies continue to define both overlapping and distinct roles for IL-13 and the related cytokine, IL-4, in promoting asthmatic changes. Furthermore, new evidence concerning the role of the "decoy" receptor, IL-13Ralpha2, has prompted re-evaluation of the receptor forms that underlie the numerous activities of IL-13. In this review, we summarize the essential role of IL-13 in asthma, compare the relative contributions of IL-13 and IL-4 to key aspects of the asthmatic phenotype, and outline novel therapeutic strategies to target this critical cytokine.
Asunto(s)
Asma/inmunología , Interleucina-13/inmunología , Animales , Asma/genética , Hiperreactividad Bronquial/inmunología , Fibrosis/inmunología , Humanos , Inflamación/inmunología , Interleucina-13/genética , Interleucina-4/inmunología , Moco/inmunología , Polimorfismo Genético , Receptores de Interleucina-13/inmunología , Receptores de Interleucina-4/inmunologíaRESUMEN
Interleukin (IL)-13 is a key cytokine driving allergic and asthmatic responses and contributes to airway inflammation in cynomolgus monkeys after segmental challenge with Ascaris suum antigen. IL-13 bioactivity is mediated by a heterodimeric receptor (IL-13Ralpha1/IL-4Ralpha) and can be inhibited in vitro by targeting IL-13 interaction with either chain. However, in cytokine systems, in vitro neutralization activity may not always predict inhibitory function in vivo. To address the efficacy of two different IL-13 neutralization mechanisms in a primate model of atopic disease, two humanized monoclonal antibodies to IL-13 were generated, with highly homologous properties, differing in epitope recognition. Ab01 blocks IL-13 interaction with IL-4Ralpha, and Ab02 blocks IL-13 interaction with IL-13Ralpha1. In a cynomolgus monkey model of IgE responses to A. suum antigen, both Ab01 and Ab02 effectively reduced serum titers of Ascaris-specific IgE and diminished ex vivo Ascaris-triggered basophil histamine release, assayed 8 weeks after a single administration of antibody. The two antibodies also produced comparable reductions in pulmonary inflammation after lung segmental challenge with Ascaris antigen. Increased serum levels of IL-13, lacking demonstrable biological activity, were seen postchallenge in animals given either anti-IL-13 antibody but not in control animals given human IgG of irrelevant specificity. These findings demonstrate a potent effect of IL-13 neutralization on IgE-mediated atopic responses in a primate system and show that IL-13 can be efficiently neutralized by targeting either the IL-4Ralpha-binding epitope or the IL-13Ralpha1-binding epitope.
Asunto(s)
Antígenos Helmínticos/inmunología , Ascaris/inmunología , Inmunoglobulina E/sangre , Inmunoglobulina G/inmunología , Inflamación/inmunología , Interleucina-13/inmunología , Pulmón/inmunología , Receptores de Interleucina-13/inmunología , Animales , Anticuerpos Antihelmínticos/inmunología , Basófilos/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Epítopos/inmunología , Liberación de Histamina/inmunología , Humanos , Macaca fascicularis , MasculinoRESUMEN
Interleukin-13 (IL-13) is a critical mediator of pulmonary pathology associated with asthma. Drugs that block the biological function of IL-13 may be an effective treatment for asthma. IL-13 signals by forming a ternary complex with IL-13Ralpha1 and IL-4R. Genetic variants of IL-13 and of its receptor components have been linked to asthma. One in particular, IL-13R110Q, is associated with increased IgE levels and asthma. We characterized the interactions of the binary complexes composed of IL-13 or IL-13R110Q with IL-13Ralpha1 and the ternary complexes composed of IL-13 or IL-13R110Q and IL-13Ralpha1 with IL-4R using surface plasmon resonance and time-resolved fluorescence resonance energy transfer (TR-FRET). By both biophysical methods, we found no differences between IL-13 and IL-13R110Q binding in either the binary or the ternary complex. IL-4R bound to the IL-13/IL-13Ralpha1 complex with slow on and off rates, resulting in a relatively weak affinity of about 100nM. We developed a TR-FRET assay targeting the interaction between the IL-4R and the binary complex. Two antibodies with known binding epitopes to IL-13 that block binding to either IL-13Ralpha1 or IL-4R inhibited the TR-FRET signal formed by the ternary complex. This assay will be useful to identify and characterize inhibitory molecules of IL-13 function.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Interleucina-13/metabolismo , Humanos , Interleucina-13/química , Subunidad alfa1 del Receptor de Interleucina-13/química , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Unión Proteica , Receptores de Interleucina-4/química , Receptores de Interleucina-4/metabolismo , Reproducibilidad de los Resultados , Resonancia por Plasmón de SuperficieRESUMEN
Protein kinase C theta (PKCtheta) is expressed in NK cells, but its functional role has not been defined. Here, we demonstrate involvement of PKCtheta in IL-12-induced NK cell IFN-gamma production. NK cells from PKCtheta(-/-) mice produced less IFN-gamma in response to IL-12 than those from wild-type (WT) mice. IL-12-induced NK cell cytotoxicity was unaffected, and NK cells from PKCtheta(-/-) mice did not display reduced IFN-gamma production in response to IL-18, indicating a specific role for PKCtheta in IL-12-induced IFN-gamma production. Under the conditions tested, T cells did not produce IFN-gamma in response to IL-12 or affect the ability of NK cells to produce the cytokine. PKCtheta deficiency did not affect NK cell numbers, granularity, viability, or cytotoxic activity in response to polyinosinic:polycytydylic acid. NK cells from PKCtheta(-/-) mice exhibited normal expression of IL-12Rbeta1 and STAT4 proteins and normal induction of STAT4 phosphorylation in response to IL-12. Phosphorylation of threonine 538 within the catalytic domain of PKCtheta was detectable in NK cells from WT mice but was not enhanced by IL-12. Transcription of IFN-gamma increased similarly in NK cells from WT and PKCtheta(-/-) mice in response to IL-12, and there was no difference in IFN-gamma mRNA stability. Taken together, these findings indicate a role for PKCtheta in the post-transcriptional regulation of IL-12-induced IFN-gamma production.
Asunto(s)
Interferón gamma/deficiencia , Interleucina-12/farmacología , Isoenzimas/genética , Células Asesinas Naturales/inmunología , Proteína Quinasa C/deficiencia , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cruzamientos Genéticos , Citocinas/farmacología , Femenino , Regulación de la Expresión Génica , Interferón gamma/biosíntesis , Interleucinas/farmacología , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa C/genética , Proteína Quinasa C-theta , Proteínas Recombinantes/farmacología , Bazo/inmunología , Linfocitos T/enzimología , Linfocitos T/inmunología , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND: Airway inflammation is a hallmark feature of asthma and a driver of airway hyperresponsiveness. IL-13 is a key inducer of airway inflammation in rodent models of respiratory disease, but a role for IL-13 has not been demonstrated in primates. OBJECTIVE: We sought to test the efficacy of a neutralizing antibody to human IL-13 in a cynomolgus monkey model of lung inflammation. METHODS: Using cynomolgus monkeys (Macaca fascicularis) that are sensitized to Ascaris suum through natural exposure, we developed a reproducible model of acute airway inflammation after segmental A suum antigen challenge. This model was used to test the in vivo efficacy of mAb13.2, a mouse mAb directed against human IL-13, and IMA-638, the humanized counterpart of mAb13.2. Bronchoalveolar lavage (BAL) cells and BAL fluid were collected before and after antigen challenge and assayed for cellular content by means of differential count. RESULTS: Total BAL cell count, eosinophil number, and neutrophil number were all reduced in animals treated with mAb13.2 or IMA-638 compared with values in control animals that were untreated, given saline, or treated with human IgG of irrelevant specificity. In addition, levels of eotaxin and RANTES in BAL fluid were reduced in anti-IL-13-treated animals compared with levels seen in control animals. CONCLUSION: These findings support a role for IL-13 in maintaining lung inflammation in response to allergen challenge in nonhuman primates. CLINICAL IMPLICATIONS: IL-13 neutralization with a specific antibody could be a useful therapeutic strategy for asthma.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Ascariasis/inmunología , Interleucina-13/antagonistas & inhibidores , Neumonía/inmunología , Neumonía/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/uso terapéutico , Antígenos Helmínticos/inmunología , Ascaris suum , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interleucina-13/genética , Interleucina-13/inmunología , Macaca fascicularis , Masculino , Datos de Secuencia Molecular , Neumonía/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
IL-13 contributes to airway hyperresponsiveness, mucus secretion, inflammation, and fibrosis, suggesting that it plays a central role in asthma pathogenesis. Neutralization of IL-13 with sIL-13Ralpha2-Fc (sIL-13R) reduces allergen-induced airway responses in rodent models of respiratory disease, but its efficacy in a large animal model has not been previously reported. In this study, we determined whether two different strategies for IL-13 neutralization modified experimental asthma in sheep. Sheep with natural airway hypersensitivity to Ascaris suum antigen were treated intravenously either with sIL-13R, a strong antagonist of sheep IL-13 bioactivity in vitro, or with IMA-638 (IgG1, kappa), a humanized antibody to human IL-13. Higher doses of IMA-638 were used because, although it is a potent antagonist of human IL-13, this antibody has 20 to 30 times lower binding and neutralization activity against sheep IL-13. Control animals received human IgG of irrelevant specificity. Sheep were treated 24 h before inhalation challenge with nebulized A. suum. The effects on antigen-induced early and late bronchial responses, and antigen-induced hyperresponsiveness, were assessed. Both sIL-13R and IMA-638 provided dose-dependent inhibition of the antigen-induced late responses and airway hyperresponsiveness. The highest dose of IMA-638 also reduced the early phase response. These findings suggest that IL-13 contributes to allergen-induced airway responses in this sheep model of asthma, and that neutralization of IL-13 is an effective strategy for blocking these A. suum-induced effects.
Asunto(s)
Asma/tratamiento farmacológico , Asma/inmunología , Modelos Animales de Enfermedad , Interleucina-13/antagonistas & inhibidores , Interleucina-13/inmunología , Oveja Doméstica/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Ascaris suum/fisiología , Asma/inducido químicamente , Asma/fisiopatología , Secuencia de Bases , Hiperreactividad Bronquial/parasitología , Hiperreactividad Bronquial/patología , Broncoconstricción/efectos de los fármacos , Broncoconstricción/inmunología , Carbacol/farmacología , Femenino , Células HT29 , Humanos , Interleucina-13/química , Interleucina-13/genética , Cinética , Datos de Secuencia Molecular , Pruebas de Neutralización , Receptores de Interleucina-13/metabolismo , Oveja Doméstica/parasitología , Solubilidad/efectos de los fármacos , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
IL-4 and IL-13 are potent cytokines that drive production of IgE, which is critical to the development of atopic disease. In this study, we directly compared IgE generation and IgE-dependent mast cell effector function in mouse strains lacking IL-4, IL-13, IL-4 + IL-13, or their common receptor component, IL-4Ralpha. Although serum IgE was undetectable under resting conditions in most animals deficient in one or both cytokines, peritoneal mast cells from mice lacking IL-4 or IL-13 had only partial reductions in surface IgE level. In contrast, peritoneal mast cells from IL-4/13(-/-) and IL-4Ralpha(-/-) animals were severely deficient in surface IgE, and showed no detectable degranulation following treatment with anti-IgE in vitro. Surprisingly, however, intradermal challenge with high concentrations of anti-IgE Ab induced an ear-swelling response in these strains, implying some capacity for IgE-mediated effector function in tissue mast cells. Furthermore, upon specific immunization with OVA, both IL-4/IL-13(-/-) and IL-4Ralpha(-/-) mice produced detectable levels of serum IgE and Ag-specific IgG1, and generated strong ear-swelling responses to intradermal administration of anti-IgE. These findings suggest that a mechanism for IgE production exists in vivo that is independent of IL-4 or IL-13.
Asunto(s)
Inmunoglobulina E/biosíntesis , Interleucina-13/deficiencia , Interleucina-13/genética , Interleucina-4/deficiencia , Interleucina-4/genética , Mastocitos/inmunología , Mastocitos/metabolismo , Animales , Sitios de Unión de Anticuerpos/genética , Recuento de Células , Degranulación de la Célula/genética , Degranulación de la Célula/inmunología , Separación Celular , Relación Dosis-Respuesta Inmunológica , Inmunización Secundaria , Inmunoglobulina E/sangre , Inmunoglobulina E/deficiencia , Inmunoglobulina E/fisiología , Interleucina-13/metabolismo , Interleucina-13/fisiología , Interleucina-4/metabolismo , Interleucina-4/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Anafilaxis Cutánea Pasiva , Cavidad Peritoneal/citología , Unión Proteica/genética , Unión Proteica/inmunología , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , Receptores de Interleucina-4/deficiencia , Receptores de Interleucina-4/genética , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
In human atopic disease, IgE sensitizes the allergic response, while IgG4 is protective. Because IL-4 and IL-13 trigger switch recombination to both IgE and IgG4, additional agents must regulate the balance between these isotypes to influence susceptibility or tolerance to atopy. In this report, we define in vitro conditions leading to activation or inhibition of human IgE and IgG4 production by IL-21. IL-21 reduced IL-4-driven IgE synthesis by mitogen-stimulated human PBMC. IL-21 inhibition of human IgE production was not a direct effect on B cells, was not seen following B cell activation with IL-13, and was overcome by CD40 ligation. Neither IFN-gamma, IL-10, IL-12, CD40L expression, nor apoptosis was responsible for the inhibitory effect. In contrast, IL-21-stimulated secretion of IgG4 from PBMC. Our findings indicate that IL-21 may influence the production of both human IgE and IgG4, and thus contribute to the regulation of atopic reactions.
Asunto(s)
Inmunoglobulina E/biosíntesis , Inmunoglobulina E/inmunología , Interleucina-13/inmunología , Interleucina-4/inmunología , Interleucinas/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Ligando de CD40/metabolismo , Proliferación Celular , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina E/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Interferón gamma/genética , Interleucina-10/biosíntesis , Interleucina-12/biosíntesis , Interleucina-13/farmacología , Interleucina-4/farmacología , Interleucinas/farmacología , Receptores de Interleucina/biosíntesis , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12RESUMEN
IFNalpha/beta, IL-12, and IL-15 regulate NK cell activation and expansion, but signals triggering resolution of the NK response upon induction of adaptive immunity remain to be defined. We now report that IL-21, a product of activated T cells, may serve this function. Mice lacking IL-21R (IL-21R(-/-)) had normal NK cell development but no detectable responses to IL-21. IL-21 enhanced cytotoxic activity and IFNgamma production by activated murine NK cells but did not support their viability, thus limiting their duration of activation. Furthermore, IL-21 blocked IL-15-induced expansion of resting NK cells, thus preventing the initiation of further innate responses. In contrast, IL-21 enhanced the proliferation, IFNgamma production, and cytotoxic function of CD8(+) effector T cells in an allogeneic MLR. These observations suggest that IL-21 promotes the transition between innate and adaptive immunity.