RESUMEN
RATIONALE: Type 2 (T2) asthma is characterized by airflow limitations and elevated levels of blood and sputum eosinophils, fractional exhaled nitric oxide, IgE, and periostin. While eosinophils are associated with exacerbations, the contribution of eosinophils to lung inflammation, remodeling and function remains largely hypothetical. OBJECTIVES: To determine the effect of T2 cytokines IL-4, IL-13 and IL-5 on eosinophil biology and compare the impact of depleting just eosinophils versus inhibiting all aspects of T2 inflammation on airway inflammation. METHODS: Human eosinophils or endothelial cells stimulated with IL-4, IL-13 or IL-5 were assessed for gene changes or chemokine release.Mice exposed to house dust mite extract received anti-IL-4Rα (dupilumab), anti-IL-5 or control antibodies and were assessed for changes in lung histological and inflammatory endpoints. MEASUREMENTS AND MAIN RESULTS: IL-4 or IL-13 stimulation of human eosinophils and endothelial cells induced gene expression changes related to granulocyte migration; whereas, IL-5 induced changes reflecting granulocyte differentiation.In a mouse model, blocking IL-4Rα improved lung function by impacting multiple effectors of inflammation and remodeling, except peripheral eosinophil counts, thereby disconnecting blood eosinophils from airway inflammation, remodeling and function. Blocking IL-5 globally reduced eosinophil counts but did not impact inflammatory or functional measures of lung pathology. Whole lung transcriptome analysis revealed that IL-5 or IL-4Rα blockade impacted eosinophil associated genes, whereas IL-4Rα blockade also impacted genes associated with multiple cells, cytokines and chemokines, mucus production, cell:cell adhesion and vascular permeability. CONCLUSIONS: Eosinophils are not the sole contributor to asthma pathophysiology or lung function decline and emphasizes the need to block additional mediators to modify lung inflammation and impact lung function.
Asunto(s)
Asma , Neumonía , Animales , Humanos , Ratones , Asma/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Endoteliales/metabolismo , Inflamación/metabolismo , Interleucina-13/metabolismo , Pulmón/metabolismo , Neumonía/metabolismo , Interleucina-4/farmacologíaRESUMEN
BACKGROUND: Blocking the major cat allergen, Fel d 1, with mAbs was effective in preventing an acute cat allergic response. OBJECTIVES: This study sought to extend the allergen-specific antibody approach and demonstrate that a combination of mAbs targeting Bet v 1, the immunodominant and most abundant allergenic protein in birch pollen, can prevent the birch allergic response. METHODS: Bet v 1-specific mAbs, REGN5713, REGN5714, and REGN5715, were isolated using the VelocImmune platform. Surface plasmon resonance, x-ray crystallography, and cryo-electron microscopy determined binding kinetics and structural data. Inhibition of IgE-binding, basophil activation, and mast cell degranulation were assessed via blocking ELISA, flow cytometry, and the passive cutaneous anaphylaxis mouse model. RESULTS: REGN5713, REGN5714, and REGN5715 bind with high affinity and noncompetitively to Bet v 1. A cocktail of all 3 antibodies, REGN5713/14/15, blocks IgE binding to Bet v 1 and inhibits Bet v 1- and birch pollen extract-induced basophil activation ex vivo and mast cell degranulation in vivo. Crystal structures of the complex of Bet v 1 with immunoglobulin antigen-binding fragments of REGN5713 or REGN5715 show distinct interaction sites on Bet v 1. Cryo-electron microscopy reveals a planar and roughly symmetrical complex formed by REGN5713/14/15 bound to Bet v 1. CONCLUSIONS: These data confirm the immunodominance of Bet v 1 in birch allergy and demonstrate blockade of the birch allergic response with REGN5713/14/15. Structural analyses show simultaneous binding of REGN5713, REGN5714, and REGN5715 with substantial areas of Bet v 1 exposed, suggesting that targeting specific epitopes is sufficient to block the allergic response.
Asunto(s)
Alérgenos/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos de Plantas/inmunología , Epítopos Inmunodominantes/inmunología , Inmunoglobulina G/farmacología , Anafilaxis Cutánea Pasiva/inmunología , Animales , Basófilos/efectos de los fármacos , Basófilos/inmunología , Humanos , Inmunoglobulina E/inmunología , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Ratones Endogámicos BALB C , Rinitis Alérgica Estacional/sangre , Rinitis Alérgica Estacional/inmunologíaRESUMEN
BACKGROUND: Severe inflammatory airway diseases are associated with inflammation that does not resolve, leading to structural changes and an overall environment primed for exacerbations. OBJECTIVE: We sought to identify and inhibit pathways that perpetuate this heightened inflammatory state because this could lead to therapies that allow for a more quiescent lung that is less predisposed to symptoms and exacerbations. METHODS: Using prolonged exposure to house dust mite in mice, we developed a mouse model of persistent and exacerbating airway disease characterized by a mixed inflammatory phenotype. RESULTS: We show that lung IL-33 drives inflammation and remodeling beyond the type 2 response classically associated with IL-33 signaling. IL-33 blockade with an IL-33 neutralizing antibody normalized established inflammation and improved remodeling of both the lung epithelium and lung parenchyma. Specifically, IL-33 blockade normalized persisting and exacerbating inflammatory end points, including eosinophilic, neutrophilic, and ST2+CD4+ T-cell infiltration. Importantly, we identified a key role for IL-33 in driving lung remodeling because anti-IL-33 also re-established the presence of ciliated cells over mucus-producing cells and decreased myofibroblast numbers, even in the context of continuous allergen exposure, resulting in improved lung function. CONCLUSION: Overall, this study shows that increased IL-33 levels drive a self-perpetuating amplification loop that maintains the lung in a state of lasting inflammation and remodeled tissue primed for exacerbations. Thus IL-33 blockade might ameliorate symptoms and prevent exacerbations by quelling persistent inflammation and airway remodeling.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Asma/inmunología , Interleucina-33/inmunología , Pulmón/inmunología , Pyroglyphidae/inmunología , Transducción de Señal/inmunología , Animales , Asma/inducido químicamente , Asma/patología , Asma/terapia , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/patología , Interleucina-33/antagonistas & inhibidores , Pulmón/patología , Ratones , Ratones Transgénicos , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Multiple sclerosis (MS) is thought to be a CD4+ T cell mediated autoimmune demyelinating disease of the central nervous system (CNS) that is rarely diagnosed during infancy. Cellular and molecular mechanisms that confer disease resistance in this age group are unknown. We tested the hypothesis that a differential composition of immune cells within the CNS modulates age-associated susceptibility to CNS autoimmune disease. C57BL/6 mice younger than eight weeks were resistant to experimental autoimmune encephalomyelitis (EAE) following active immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p) 35-55. Neonates also developed milder EAE after transfer of adult encephalitogenic T cells primed by adult or neonate antigen presenting cells (APC). There was a significant increase in CD45+ hematopoietic immune cells and CD45+ high side scatter granulocytes in the CNS of adults, but not in neonates. Within the CD45+ immune cell compartment of adults, the accumulation of CD4+ T cells, Gr-1+ and Gr-1- monocytes and CD11c+ dendritic cells (DC) was identified. A significantly greater percentage of CD19+ B cells in the adult CNS expressed MHC II than neonate CNS B cells. Only in the adult CNS could IFNγ transcripts be detected 10 days post immunization for EAE. IFNγ is highly expressed by adult donor CD4+ T cells that are adoptively transferred but not by transferred neonate donor cells. In contrast, IL-17 transcripts could not be detected in adult or neonate CNS in this EAE model, and neither adult nor neonate donor CD4+ T cells expressed IL-17 at the time of adoptive transfer.
Asunto(s)
Linfocitos B/patología , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/patología , Células TH1/patología , Traslado Adoptivo , Animales , Animales Recién Nacidos , Diferenciación Celular/fisiología , Proliferación Celular , Citometría de Flujo , Genes MHC Clase II/genética , Antígeno Ki-67/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Glicoproteína Mielina-Oligodendrócito/metabolismo , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Linfocitos T Colaboradores-Inductores/fisiologíaRESUMEN
Immunoglobulin E (IgE) is a key driver of type 1 hypersensitivity reactions and allergic disorders, which are globally increasing in number and severity. Although eliminating pathogenic IgE may be a powerful way to treat allergy, no therapeutic strategy reported to date can fully ablate IgE production. Interleukin-4 receptor α (IL-4Rα) signaling is required for IgE class switching, and IL-4Rα blockade gradually reduces, but does not eliminate, IgE. The persistence of IgE after IL-4Rα blockade may be due to long-lived IgE+ plasma cells that maintain serological memory to allergens and thus may be susceptible to plasma cell-targeted therapeutics. We demonstrate that transient administration of a B cell maturation antigen x CD3 (BCMAxCD3) bispecific antibody markedly depletes IgE, as well as other immunoglobulins, by ablating long-lived plasma cells, although IgE and other immunoglobulins rapidly rebound after treatment. Concomitant IL-4Rα blockade specifically and durably prevents the reemergence of IgE by blocking IgE class switching while allowing the restoration of other immunoglobulins. Moreover, this combination treatment prevented anaphylaxis in mice. Together with additional cynomolgus monkey and human data, our studies demonstrate that allergic memory is primarily maintained by both non-IgE+ memory B cells that require class switching and long-lived IgE+ plasma cells. Our combination approach to durably eliminate pathogenic IgE has potential to benefit allergy in humans while preserving antibody-mediated immunity.
Asunto(s)
Anafilaxia , Inmunoglobulina E , Ratones , Humanos , Animales , Macaca fascicularis , Células Plasmáticas , AlérgenosRESUMEN
The common γ chain (γc; IL-2RG) is a subunit of the interleukin (IL) receptors for the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The lack of appropriate neutralizing antibodies recognizing IL-2RG has made it difficult to thoroughly interrogate the role of γc cytokines in inflammatory and autoimmune disease settings. Here, we generated a γc cytokine receptor antibody, REGN7257, to determine whether γc cytokines might be targeted for T cell-mediated disease prevention and treatment. Biochemical, structural, and in vitro analysis showed that REGN7257 binds with high affinity to IL-2RG and potently blocks signaling of all γc cytokines. In nonhuman primates, REGN7257 efficiently suppressed T cells without affecting granulocytes, platelets, or red blood cells. Using REGN7257, we showed that γc cytokines drive T cell-mediated disease in mouse models of graft-versus-host disease (GVHD) and multiple sclerosis by affecting multiple aspects of the pathogenic response. We found that our xenogeneic GVHD mouse model recapitulates hallmarks of acute and chronic GVHD, with T cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. Our findings indicate that γc cytokines contribute to GVHD and aplastic anemia pathology by promoting these characteristic features. By demonstrating that broad inhibition of γc cytokine signaling with REGN7257 protects from immune-mediated disorders, our data provide evidence of γc cytokines as key drivers of pathogenic T cell responses, offering a potential strategy for the management of T cell-mediated diseases.
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Anemia Aplásica , Enfermedad Injerto contra Huésped , Subunidad gamma Común de Receptores de Interleucina , Linfocitos T , Animales , Ratones , Anemia Aplásica/metabolismo , Anticuerpos Monoclonales/metabolismo , Citocinas/metabolismo , Enfermedad Injerto contra Huésped/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/patología , Subunidad gamma Común de Receptores de Interleucina/antagonistas & inhibidores , Subunidad gamma Común de Receptores de Interleucina/metabolismo , PrimatesRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is a relevant animal model for the human demyelinating inflammatory disorder of the central nervous system (CNS), multiple sclerosis (MS). Induction of EAE by adoptive transfer allows studying the role of the donor T lymphocyte in disease pathogenesis. It has been challenging to reliably induce adoptive transfer EAE in C57BL/6 (H-2b) mice. The goal of this study was to develop a reproducible and high yield protocol for adoptive transfer EAE in C57BL/6 mice. A step-wise experimental approach permitted us to develop a protocol that resulted in a consistent relatively high disease incidence of ~70% in recipient mice. Donor mice were immunized with myelin oligodendrocyte glycoprotein (MOG)p35-55 in complete Freund's adjuvant (CFA) followed by pertussis toxin (PT). Only lymph node cells (LNC) isolated at day 12 post immunization, and restimulated in vitro for 72 hours with 10 µg/mL of MOGp35-55 and 0.5 ng/mL of interleukin-12 (IL-12) were able to transfer disease. The ability of LNC to transfer disease was associated with the presence of inflammatory infiltrates in the CNS at day 12. Interferon gamma (IFNγ) was produced at comparable levels in cell cultures prepared from mice at both day 6 and day 12 post immunization. By contrast, there was a trend towards a negative association between IL-17 and disease susceptibility in our EAE model. The amount of GM-CSF secreted was significantly increased in the culture supernatants from cells collected at day 12 post immunization versus those collected at day 6 post-immunization. Activated CD4+ T cells present in the day 12 LNC cultures maintained expression of the transcription factor T-bet, which has been shown to regulate the expression of the IL-23 receptor. Also, there was an increased prevalence of MOGp35-55-specific CD4+ T cells in day 12 LNC after in vitro re-stimulation. In summary, encephalitogenic LNC that adoptively transfer EAE in C57BL/6 mice were not characterized by a single biomarker in our study, but by a composite of inflammatory markers. Our data further suggest that GM-CSF expression by CD4+ T cells regulated by IL-23 contributes to their encephalitogenicity in our EAE model.
Asunto(s)
Traslado Adoptivo/métodos , Linfocitos T CD4-Positivos/fisiología , Encefalomielitis Autoinmune Experimental/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Ganglios Linfáticos/citología , Proteínas de Dominio T Box/inmunología , Animales , Biomarcadores/metabolismo , Encéfalo/inmunología , Encéfalo/patología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/fisiopatología , Glicoproteínas/administración & dosificación , Glicoproteínas/inmunología , Humanos , Interferón gamma/inmunología , Interleucina-12/inmunología , Interleucina-17/inmunología , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Médula Espinal/inmunología , Médula Espinal/patologíaRESUMEN
OBJECTIVE: To determine if suppressing Nogo-A, an axonal inhibitory protein, will promote functional recovery in a murine model of multiple sclerosis (MS). METHODS: A small interfering RNA was developed to specifically suppress Nogo-A (siRNA-NogoA). The siRNA-NogoA silencing effect was evaluated in vitro and in vivo via immunohistochemistry. The siRNA was administered intravenously in 2 models of experimental autoimmune encephalomyelitis (EAE). Axonal repair was measured by upregulation of GAP43. Enzyme-linked immunosorbent assay, flow cytometry, and (3)H-thymidine incorporation were used to determine immunological changes in myelin-specific T cells in mice with EAE. RESULTS: The siRNA-NogoA suppressed Nogo-A expression in vitro and in vivo. Systemic administration of siRNA-NogoA ameliorated EAE and promoted axonal repair, as demonstrated by enhanced GAP43+ axons in the lesions. Myelin-specific T-cell proliferation and cytokine production were unchanged in the siRNA-NogoA-treated mice. INTERPRETATION: Silencing Nogo-A in EAE promotes functional recovery. The therapeutic benefit appears to be mediated by axonal growth and repair, and is not attributable to changes in the encephalitogenic capacity of the myelin-specific T cells. Silencing Nogo-A may be a therapeutic option for MS patients to prevent permanent functional deficits caused by immune-mediated axonal damage.
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Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Proteínas de la Mielina/metabolismo , ARN Interferente Pequeño/uso terapéutico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo/métodos , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glicoproteínas/efectos adversos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/farmacología , Proteínas de la Mielina/genética , Glicoproteína Mielina-Oligodendrócito , Neuroblastoma , Proteínas Nogo , Fragmentos de Péptidos/efectos adversos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , ARN Interferente Pequeño/genética , Médula Espinal/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Transfección/métodosRESUMEN
Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists have been shown to have a therapeutic benefit in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). In this study, we investigated the mechanism by which the PPARalpha agonist gemfibrozil induces immune deviation and protects mice from EAE. We demonstrated that treatment with gemfibrozil increases expression of the Th2 transcription factor GATA-3 and decreases expression of the Th1 transcription factor T-bet in vitro and directly ex vivo. These changes correlated with an increase in nuclear PPARalpha expression. Moreover, the protective effects of PPARalpha agonists in EAE were shown to be partially dependent on IL-4 and to occur in a receptor-dependent manner. PPARalpha was demonstrated, for the first time, to regulate the IL-4 and IL-5 genes and to bind the IL-4 promoter in the presence of steroid receptor coactivator-1, indicating that PPARalpha can directly transactivate the IL-4 gene. Finally, therapeutic administration of PPARalpha agonists ameliorated clinically established EAE, suggesting that PPARalpha agonists may provide a treatment option for immune-mediated inflammatory diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Gemfibrozilo/farmacología , Factores Inmunológicos/farmacología , PPAR alfa/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Animales , Western Blotting , Citocinas/biosíntesis , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/genética , Ensayo de Inmunoadsorción Enzimática , Fenofibrato/farmacología , Factor de Transcripción GATA3/biosíntesis , Factor de Transcripción GATA3/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-5/genética , Interleucina-5/metabolismo , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/efectos de los fármacos , TransfecciónRESUMEN
The primary biological function of the endogenous cellular prion protein has remained unclear. We investigated its biological function in the generation of cellular immune responses using cellular prion protein gene-specific small interfering ribonucleic acid in vivo and in vitro. Our results were confirmed by blocking cellular prion protein with monovalent antibodies and by using cellular prion protein-deficient and -transgenic mice. In vivo prion protein gene-small interfering ribonucleic acid treatment effects were of limited duration, restricted to secondary lymphoid organs and resulted in a 70% reduction of cellular prion protein expression in leukocytes. Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity. In vivo prion protein gene-small interfering ribonucleic acid treatment promoted T cell differentiation towards pro-inflammatory phenotypes and increased survival of antigen-specific T cells. Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis. Finally, treatment of myelin basic protein(1-11) T cell receptor transgenic mice with prion protein gene-small interfering ribonucleic acid resulted in spontaneous experimental autoimmune encephalomyelitis. Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses. Our findings indicate that cellular prion protein regulates T cell receptor-mediated T cell activation, differentiation and survival. Defects in autoimmunity are restricted to the immune system and not the central nervous system. Our data identify cellular prion protein as a regulator of cellular immunological homoeostasis and suggest cellular prion protein as a novel potential target for therapeutic immunomodulation.
Asunto(s)
Enfermedades Autoinmunes Desmielinizantes SNC/genética , Silenciador del Gen/inmunología , Priones/genética , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/inmunología , Animales , Enfermedades Autoinmunes Desmielinizantes SNC/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Priones/inmunología , ARN Interferente Pequeño/genéticaRESUMEN
Immunoglobulin E (IgE) plays an important role in allergic diseases. Nevertheless, the source of IgE serological memory remains controversial. We reexamined the mechanism of serological memory in allergy using a dual reporter system to track IgE+ plasma cells in mice. Short-term allergen exposure resulted in the generation of IgE+ plasma cells that resided mainly in secondary lymphoid organs and produced IgE that was unable to degranulate mast cells. In contrast, chronic allergen exposure led to the generation of long-lived IgE+ plasma cells that were primarily derived from sequential class switching of IgG1, accumulated in the bone marrow, and produced IgE capable of inducing anaphylaxis. IgE+ plasma cells were found in the bone marrow of human allergic, but not nonallergic donors, and allergen-specific IgE produced by these cells was able to induce mast cell degranulation when transferred to mice. These data demonstrate that long-lived IgE+ bone marrow plasma cells arise during chronic allergen exposure and establish serological memory in both mice and humans.
Asunto(s)
Alérgenos/inmunología , Inmunoglobulina E/sangre , Memoria Inmunológica , Células Plasmáticas/inmunología , Pyroglyphidae/inmunología , Anafilaxia/inmunología , Animales , Células de la Médula Ósea/inmunología , Exposición a Riesgos Ambientales , Humanos , Mastocitos/inmunología , RatonesRESUMEN
BACKGROUND: Interleukin (IL)-12 and IL-23 are heterodimers that share the p40 subunit, and both cytokines are critical in the differentiation of T helper (Th)1 and Th17 cells, respectively. Th1 and Th17 effector cells have been implicated in the pathogenesis of experimental autoimmune encephalitis (EAE), an animal model of the human central nervous system (CNS) autoimmune demyelinating disorder multiple sclerosis (MS). However, ustekinumab, a monoclonal antibody (mAb) against p40 failed to show efficacy over placebo in a phase II clinical trial in patients with MS. The role of p40 in initial T cell priming and maintenance in secondary lymphoid tissues is not yet well understood. METHODS: Active EAE was induced in the B6.129-IL12b strain of p40eYFP reporter mice (yet40 mice), and Th1 and Th17 polarized cells were adoptively transferred into p40-deficient mice. Cellular subsets were phenotyped by multi-parameter flow cytometry, and p40 tissue expression was identified by confocal microscopy. RESULTS: We show that yet40 mice are susceptible to EAE, and that p40 is highly expressed in secondary lymphoid organs and the CNS during all stages of the disease. Interestingly, p40 expression in the recipient is not required for EAE induction after adoptive transfer of activated and differentiated encephalitogenic Th1 and Th17 cells into p40-deficient mice. Peripheral antagonism of T helper cell trophic factors critical for the differentiation and maintenance of Th1 and Th17 cells ameliorates EAE, indicating that p40 may play a critical role in the induction of CNS autoimmunity but not in its perpetuation. CONCLUSION: Our data may explain why ustekinumab did not ameliorate paraclinical and clinical disease in patients with MS. In patients with already established disease, activated antigen-specific encephalitogenic CD4+ T cells are likely already differentiated, and are not dependent on p40 for maintenance. A clinical trial of longer duration with anti-p40 mAbs or other forms of pharmacological p40 antagonism, or sequential anti-p40 therapy following T cell depletion may show a benefit by affecting de novo generation of autoimmune T cells.
Asunto(s)
Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Subunidad p40 de la Interleucina-12/metabolismo , Ganglios Linfáticos/inmunología , Bazo/inmunología , Traslado Adoptivo/métodos , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Ratones , Células TH1/inmunología , Células TH1/trasplante , Células Th17/inmunología , Células Th17/trasplante , Regulación hacia ArribaRESUMEN
Recent clinical trials have established B cell depletion by the anti-CD20 chimeric antibody Rituximab as a beneficial therapy for patients with relapsing-remitting multiple sclerosis (MS). The impact of Rituximab on T cell responses remains largely unexplored. In the experimental autoimmune encephalomyelitis (EAE) model of MS in mice that express human CD20, Rituximab administration rapidly depleted peripheral B cells and strongly reduced EAE severity. B cell depletion was also associated with diminished Delayed Type Hypersensitivity (DTH) and a reduction in T cell proliferation and IL-17 production during recall immune response experiments. While Rituximab is not considered a broad immunosuppressant, our results indicate a role for B cells as a therapeutic cellular target in regulating encephalitogenic T cell responses in specific tissues.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Autoinmunidad/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Inmunidad Celular/efectos de los fármacos , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Autoinmunidad/inmunología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Evaluación Preclínica de Medicamentos , Encefalomielitis Autoinmune Experimental/complicaciones , Encefalomielitis Autoinmune Experimental/patología , Factores Inmunológicos/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Esclerosis Múltiple Recurrente-Remitente/complicaciones , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/inmunología , Esclerosis Múltiple Recurrente-Remitente/patología , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/inmunología , Rituximab , Linfocitos T/efectos de los fármacosRESUMEN
BACKGROUND: Minocycline is an oral tetracycline derivative with good bioavailability in the central nervous system (CNS). Minocycline, a potent inhibitor of matrix metalloproteinase (MMP)-9, attenuates disease activity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Potential adverse effects associated with long-term daily minocycline therapy in human patients are concerning. Here, we investigated whether less frequent treatment with long-circulating polyethylene glycol (PEG) minocycline liposomes are effective in treating EAE. FINDINGS: Performing in vitro time kinetic studies of PEG minocycline-liposomes in human peripheral blood mononuclear cells (PBMCs), we determined that PEG minocycline-liposome preparations stabilized with CaCl(2) are effective in diminishing MMP-9 activity. Intravenous injections of PEG minocycline-liposomes every five days were as effective in ameliorating clinical EAE as daily intraperitoneal injections of minocycline. Treatment of animals with PEG minocycline-liposomes significantly reduced the number of CNS-infiltrating leukocytes, and the overall expression of MMP-9 in the CNS. There was also a significant suppression of MMP-9 expression and proteolytic activity in splenocytes of treated animals, but not in CNS-infiltrating leukocytes. Thus, leukocytes gaining access to the brain and spinal cord require the same absolute amount of MMP-9 in all treatment groups, but minocycline decreases the absolute cell number. CONCLUSIONS: Our data indicate that less frequent injections of PEG minocycline-liposomes are an effective alternative pharmacotherapy to daily minocycline injections for the treatment of CNS autoimmune diseases. Also, inhibition of MMP-9 remains a promising treatment target in EAE and patients with MS.
Asunto(s)
Antibacterianos/administración & dosificación , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Minociclina/administración & dosificación , Polietilenglicoles/metabolismo , Animales , Antibacterianos/uso terapéutico , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Humanos , Liposomas , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Minociclina/uso terapéutico , Enfermedad Autoinmune Experimental del Sistema Nervioso/tratamiento farmacológico , Enfermedad Autoinmune Experimental del Sistema Nervioso/inmunología , Polietilenglicoles/farmacologíaRESUMEN
IL-17-producing T cells (Th17) have recently been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. However, little is known about the transcription factors that regulate these cells. Although it is clear that the transcription factor T-bet plays an essential role in the differentiation of IFN-gamma-producing CD4(+) Th1 lymphocytes, the potential role of T-bet in the differentiation of Th17 cells is not completely understood. In this study, therapeutic administration of a small interfering RNA specific for T-bet significantly improved the clinical course of established EAE. The improved clinical course was associated with suppression of newly differentiated T cells that express IL-17 in the CNS as well as suppression of myelin basic protein-specific Th1 autoreactive T cells. Moreover, T-bet was found to directly regulate transcription of the IL-23R, and, in doing so, influenced the fate of Th17 cells, which depend on optimal IL-23 production for survival. We now show for the first time that suppression of T-bet ameliorates EAE by limiting the differentiation of autoreactive Th1 cells, as well as inhibiting pathogenic Th17 cells via regulation of IL-23R.
Asunto(s)
Autoinmunidad , Interleucina-17 , Interleucina-23/genética , Proteínas de Dominio T Box/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Células TH1/inmunología , Animales , Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/inmunología , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Proteína Básica de Mielina , Proteínas del Tejido Nervioso/inmunología , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , Proteínas de Dominio T Box/inmunología , Subgrupos de Linfocitos T/inmunología , Factores de Transcripción/inmunologíaRESUMEN
The mechanisms of T cell vaccination (TCV) are still unclear, especially the molecular interactions for recognition of autoreactive T cells by the immune system. Here we investigated the role of CD28:B7 interaction in TCV-induced protection in the murine EAE model. We demonstrate that there is increased expression of both B7-1 and B7-2 on autoreactive Th1 cells compared to Th2 cells. Blockade of B7 on the vaccinating autoreactive T cell surface or blockade of CD28 in recipient mice reduced the protective effect of TCV. Furthermore, we showed that TCV significantly inhibited Ag-specific CD4 and CD8 T cell proliferation and decreased Ag-specific IFN-gamma production by CD4 T cells in mice undergoing TCV, and blocking of B7 on the surface of vaccinating T cells reduced this inhibition on Ag-specific CD4 and CD8 T cell proliferation, more significantly on Ag-specific CD4 T cell proliferation. These data indicated that B7 expression on autoreactive T cells is necessary for the recognition of autoreactive T cells by the immune system and subsequent protection from EAE in mice undergoing TCV.
Asunto(s)
Antígeno B7-1/inmunología , Antígenos CD28/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Células TH1/inmunología , Células Th2/inmunología , Vacunación , Animales , Autoinmunidad , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Ratones , Ratones Transgénicos , Células TH1/trasplante , Células Th2/trasplanteRESUMEN
In clinical transplantation host CTL are major effectors of acute rejection, and graft endothelial cells (EC) are major targets of the CTL response. It is unclear what roles CTL will play in pig-into-human xenotransplantation. We compared the mechanisms of killing used by human CTL (huCTL) vs allogeneic and pig xenogeneic EC targets. Both responses show MHC class I restriction of target cell recognition. A granzyme B inhibitor peptide completely blocks anti-human and partially blocks anti-pig responses, while inhibitory Fas ligand Ab only blocks killing of porcine cells despite similar levels of Fas expression in both target cell types. Transduction of Bcl-2 completely protects human EC from huCTL, but has no effect on huCTL-mediated killing of porcine EC despite its efficacy vs drug-induced apoptosis. Bcl-2 effectively protects human EC rendered sensitive to Fas ligand by overexpressing Fas from huCTL, yet fails to protect porcine aortic endothelial cells from huCTL in the presence of anti-Fas ligand Ab. These data reveal differences in the susceptibility of human and porcine targets to huCTL.