RESUMO
Patients with severe Th2 type asthma often have a steroid resistant phenotype and are prone to acute exacerbations. Current novel therapies have only marginal therapeutic effects. One of the hypotheses for lack of major efficacy in most patients is targeting only one redundant pathway leaving others active. Hence, we have designed and developed novel highly potent bispecific anti-TSLP/IL13 antibodies called Zweimabs (monovalent bispecific) and Doppelmabs (bivalent bispecific) that concurrently inhibits the signaling by these two cytokines.
Assuntos
Anticorpos Biespecíficos/química , Citocinas/imunologia , Interleucina-13/imunologia , Anticorpos Monoclonais/química , Células Cultivadas , Citocinas/química , Mapeamento de Epitopos , Humanos , Interleucina-13/química , Linfopoietina do Estroma do TimoRESUMO
The past 20 years have seen a proliferation of scientific data on the pathophysiology of asthma. Most of these data were generated in mice using tool reagents, gene-deficient or transgenic animals. In contrast, studies on disease pathogenesis in patients are scarce. Previously, a good novel antiasthma target for drug development was one that abrogated asthma in mice when it was knocked out, neutralized or induced asthma when it was overexpressed. This type of approach led to many drug candidates that worked in mice but unfortunately failed in patients, thereby demonstrating that the results of experiments in mice are not always predictive of clinical efficacy. Currently, there is active debate about the use of mouse models in drug discovery. In this review, we summarize the obstacles and challenges faced when using experimental mouse models of asthma in drug discovery. We propose that the initial selection of a novel drug target begins with defining the unmet medical need and specific patient population, followed by a thorough evaluation of available human data, and, only then, well-planned and executed mouse asthma experiments. Using this approach, we argue that mouse models lend support for the target when the models are tailored for the specific asthma patient population, and that targeted, reliable, and predictive mouse models can indeed improve and accelerate the drug discovery process.
Assuntos
Antiasmáticos , Modelos Animais de Doenças , Descoberta de Drogas , Animais , Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Humanos , CamundongosRESUMO
Smoking is an important risk factor for the development of chronic obstructive pulmonary disease (COPD) and viral infections are believed to be major triggers of exacerbations, which periodically lead to a worsening of symptoms. The pro-inflammatory IL-1 family members IL-1α and IL-1ß are increased in COPD patients and might contribute to disease pathology. We investigated whether individual or combined inhibition of these cytokines reduced lung inflammation in cigarette smoke (CS)-exposed and H1N1-infected BALB/c mice. Animals were treated with individual or combined antibodies (Abs) directed against IL-1α, IL-1ß or IL-1R1. Cells in BAL fluid and cytokines/chemokines in lung homogenate were determined. The viral load was investigated. Blocking IL-1α had significant suppressive effects on total cells, neutrophils, and macrophages. Furthermore, it reduced KC levels significantly. Blocking of IL-1ß did not provide significant activity. In primary human bronchial epithelial air-liquid-interface cell cultures infected with H1N1, IL-1α Abs but not IL-1ß Abs reduced levels of TNF-α and IL-6. Concomitant usage of Abs against IL-1α/IL-1ß revealed strong effects in vivo and reduced total cells, neutrophils and macrophages. Additionally, levels of KC, IL-6, TNF-α, MCP-1, MIP-1α and MIP-1ß were significantly reduced and ICAM-1 and MUC5 A/C mRNA expression was attenuated. The viral load decreased significantly upon combined IL-1α/IL-1ß Ab treatment. Blocking the IL-1R1 provided significant effects on total cells, neutrophils and macrophages but was inferior compared to inhibiting both its soluble ligands IL-1α/IL-1ß. Our results suggest that combined inhibition of IL-1α/IL-1ß might be beneficial to reduce CS/H1N1-induced airway inflammation. Moreover, combined targeting of both IL-1α/IL-1ß might be more efficient compared to individual neutralization IL-1α or IL-1ß or inhibition of the IL-1R1.
Assuntos
Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Pneumonia/prevenção & controle , Fumar/efeitos adversos , Animais , Anticorpos , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/etiologia , Inflamação/patologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/complicações , Interleucina-1alfa/imunologia , Interleucina-1beta/imunologia , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/metabolismo , Infecções por Orthomyxoviridae/complicações , Pneumonia/etiologia , Fatores de Risco , Fumaça/efeitos adversos , Nicotiana , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BI 1002494 [(R)-4-{(R)-1-[7-(3,4,5-trimethoxy-phenyl)-[1,6]napthyridin-5-yloxy]-ethyl}pyrrolidin-2-one] is a novel, potent, and selective spleen tyrosine kinase (SYK) inhibitor with sustained plasma exposure after oral administration in rats, which qualifies this molecule as a good in vitro and in vivo tool compound. BI 1002494 exhibits higher potency in inhibiting high-affinity IgE receptor-mediated mast cell and basophil degranulation (IC50 = 115 nM) compared with B-cell receptor-mediated activation of B cells (IC50 = 810 nM). This may be explained by lower kinase potency when the physiologic ligand B-cell linker was used, suggesting that SYK inhibitors may exhibit differential potency depending on the cell type and the respective signal transduction ligand. A 3-fold decrease in potency was observed in rat basophils (IC50 = 323 nM) compared with human basophils, but a similar species potency shift was not observed in B cells. The lower potency in rat basophils was confirmed in both ex vivo inhibition of bronchoconstriction in precision-cut rat lung slices and in reversal of anaphylaxis-driven airway resistance in rats. The different cellular potencies translated into different in vivo efficacy; full efficacy in a rat ovalbumin model (that contains an element of mast cell dependence) was achieved with a trough plasma concentration of 340 nM, whereas full efficacy in a rat collagen-induced arthritis model (that contains an element of B-cell dependence) was achieved with a trough plasma concentration of 1400 nM. Taken together, these data provide a platform from which different estimates of human efficacious exposures can be made according to the relevant cell type for the indication intended to be treated.
Assuntos
Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Basófilos/efeitos dos fármacos , Basófilos/enzimologia , Naftiridinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirrolidinas/farmacologia , Pirrolidinonas/farmacologia , Quinase Syk/antagonistas & inibidores , Administração Oral , Animais , Humanos , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/enzimologia , Naftiridinas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Pirrolidinas/administração & dosagem , Pirrolidinonas/administração & dosagem , RatosRESUMO
Viral infections trigger exacerbations in chronic obstructive pulmonary disease (COPD), and tiotropium, a M3 receptor antagonist, reduces exacerbations in patients by unknown mechanisms. In this report, we investigated whether tiotropium has anti-inflammatory effects in mice exposed to cigarette smoke (CS) and infected with influenza virus A/PR/8/34 (H1N1) or respiratory syncytial virus (RSV) and compared these effects with those of steroid fluticasone and PDE4-inhibitor roflumilast. Mice were exposed to CS; infected with H1N1 or RSV; and treated with tiotropium, fluticasone, or roflumilast. The amount of cells and cytokine levels in the airways, lung function, and viral load was determined. NCI-H292 cells were infected with H1N1 or RSV and treated with the drugs. In CS/H1N1-exposed mice, tiotropium reduced neutrophil and macrophage numbers and levels of interleukin-6 (IL-6) and interferon-γ (IFN-γ) in the airways and improved lung function. In contrast, fluticasone increased the loss of body weight; failed to reduce neutrophil or macrophage numbers; increased IL-6, KC, and tumor necrosis factor-α (TNF-α) in the lungs; and worsened lung function. Treatment with roflumilast reduced macrophage numbers, IL-6, and KC in the lungs but had no effect on neutrophil numbers or lung function. In CS/RSV-exposed mice, treatment with tiotropium, but not fluticasone or roflumilast, reduced neutrophil numbers and IL-6 and TNF-α levels in the lungs. Viral load of H1N1 and RSV was significantly elevated in CS/virus-exposed mice and NCI-H292 cells after fluticasone treatment, whereas tiotropium and roflumilast had no effect. In conclusion, tiotropium has anti-inflammatory effects on CS/virus-induced inflammation in mice that are superior to the effects of roflumilast and fluticasone. This finding might help to explain the observed reduction of exacerbation rates in COPD patients.
Assuntos
Anti-Inflamatórios/farmacologia , Nicotiana/química , Pneumonia/induzido quimicamente , Pneumonia/virologia , Fumaça/efeitos adversos , Brometo de Tiotrópio/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Peso Corporal/efeitos dos fármacos , Contagem de Células , Colina O-Acetiltransferase/genética , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Vírus da Influenza A Subtipo H1N1/fisiologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Oxo-Ácido-Liases/genética , Pneumonia/tratamento farmacológico , Pneumonia/patologia , Vírus Sinciciais Respiratórios/fisiologia , Brometo de Tiotrópio/uso terapêuticoRESUMO
Viral vectors have been applied successfully to generate disease-related animal models and to functionally characterize target genes in vivo. However, broader application is still limited by complex vector production, biosafety requirements, and vector-mediated immunogenic responses, possibly interfering with disease-relevant pathways. Here, we describe adeno-associated virus (AAV) variant 6.2 as an ideal vector for lung delivery in mice, overcoming most of the aforementioned limitations. In a proof-of-concept study using AAV6.2 vectors expressing IL-13 and transforming growth factor-ß1 (TGF-ß1), we were able to induce hallmarks of severe asthma and pulmonary fibrosis, respectively. Phenotypic characterization and deep sequencing analysis of the AAV-IL-13 asthma model revealed a characteristic disease signature. Furthermore, suitability of the model for compound testing was also demonstrated by pharmacological intervention studies using an anti-IL-13 antibody and dexamethasone. Similarly, the AAV-TGF-ß1 fibrosis model showed several disease-like pathophenotypes monitored by micro-computed tomography imaging and lung function measurement. Most importantly, analyses using stuffer control vectors demonstrated that in contrast to a common adenovirus-5 vector, AAV6.2 vectors did not induce any measurable inflammation and therefore carry a lower risk of altering relevant readouts. In conclusion, we propose AAV6.2 as an ideal vector system for the functional characterization of target genes in the context of pulmonary diseases in mice.
Assuntos
Asma/imunologia , Dependovirus/genética , Fibrose Pulmonar Idiopática/imunologia , Animais , Asma/genética , Asma/metabolismo , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Interleucina-13/biossíntese , Interleucina-13/genética , Camundongos Endogâmicos BALB C , Transdução Genética , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genéticaRESUMO
Severe asthma is characterised by persistent inflammation, hyperreactivity and remodeling of the airways. No efficient treatment is available, this is particularly the case for steroid resistant phenotypes. Our aim therefore was to develop a preclinical model showing characteristics of severe human asthma including steroid insensitivity. Mice were first sensitized with ovalbumin, extracts of cockroach or house dust mite followed by a challenge period of seven weeks. Further to this, an additional group of mice was sensitized with all three allergens and then challenged with allergen alternating weekly between allergens. All three allergens applied separately to the mice induced comparably strong Th2-type airway inflammation, airway hyperreactivity and airway remodeling, which was characterised by fibrosis and increased smooth muscle thickness. In contrast, application of all three allergens together resulted in a greater Th2 response and increased airway hyperreactivity and a stronger albeit not significant remodeling phenotype compared to using HDM or CRA. In this triple allergen model dexamethasone application, during the last 4 weeks of challenge, showed no suppressive effects on any of these parameters in this model. In contrast, both TLR7 agonist resiquimod and TLR9 agonist CpG-ODN reduced allergen-specific IgE, eosinophils, and collagen I in the lungs. The TLR9 agonist also reduced IL-4 and IL-5 whilst increasing IFN-γ and strongly IL-10 levels in the lungs, effects not seen with the TLR7 agonist. However, neither TLR agonist had any effect on airway hyperreactivity and airway smooth muscle mass. In conclusion we have developed a severe asthma model, which is steroid resistant and only partially sensitive to TLR7 and TLR9 agonist treatment. This model may be particular useful to test new potential therapeutics aiming at treating steroid resistant asthma in humans and investigating the underlying mechanisms responsible for steroid insensitivity.
Assuntos
Alérgenos/imunologia , Asma/imunologia , Asma/metabolismo , Dexametasona/farmacologia , Resistência a Medicamentos , Receptor 7 Toll-Like/agonistas , Receptor Toll-Like 9/agonistas , Remodelação das Vias Aéreas/imunologia , Alérgenos/administração & dosagem , Animais , Asma/tratamento farmacológico , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/metabolismo , Dexametasona/administração & dosagem , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Imunoglobulina E/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Ovalbumina/efeitos adversos , Ovalbumina/imunologia , Fenótipo , Células Th2/imunologia , Células Th2/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismoRESUMO
Recurrent relapses of allergic lung inflammation in asthmatics may lead to airway remodeling and lung damage. We tested the efficacy of tiotropium bromide, a selective long-acting, muscarinic receptor antagonist as an adjunct therapy in relapses of allergic asthma in mice. We compared the effectiveness of local intranasal administration of tiotropium and dexamethasone in acute and relapsing allergic asthma in BALB/c mice. Although tiotropium at low doses is a potent bronchodilator, we tested higher doses to determine effectiveness on inflammation and mucus hypersecretion. A 5-day course of twice daily intranasal tiotropium or dexamethasone (1 mg/kg (b.w.)) suppressed airway eosinophils by over 87% during disease initiation and 88% at relapse compared to vehicle alone. Both drugs were comparable in their capacity to suppress airway and parenchymal inflammation and mucus hypersecretion, though tiotropium was better than dexamethasone at reducing mucus secretion during disease relapse. Despite treatment with either drug, serum antigen-specific IgE or IgG1 antibody titres remained unchanged. Our study indicates that tiotropium at higher doses than required for bronchodilation, effectively suppresses inflammation and mucus hypersecretion in the lungs and airways of mice during the initiation and relapse of asthma. Tiotropium is currently not approved for use in asthma. Clinical studies have to demonstrate the efficacy of tiotropium in this respiratory disease.
Assuntos
Asma/tratamento farmacológico , Broncodilatadores/farmacologia , Dexametasona/farmacologia , Derivados da Escopolamina/farmacologia , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacologia , Asma/imunologia , Asma/fisiopatologia , Broncodilatadores/administração & dosagem , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eosinófilos/metabolismo , Feminino , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Muco/metabolismo , Recidiva , Derivados da Escopolamina/administração & dosagem , Brometo de TiotrópioRESUMO
The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4(-/-) and Mrp4(+/+) mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4(-/-) and Mrp4(+/+) mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4(+/+) or Mrp4(-/-) mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.
Assuntos
Alérgenos/imunologia , Eosinófilos/imunologia , Lipopolissacarídeos/farmacologia , Pulmão/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Neutrófilos/imunologia , Fumar/efeitos adversos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Asma/imunologia , Asma/metabolismo , Líquido da Lavagem Broncoalveolar , AMP Cíclico/sangue , Citocinas/metabolismo , Dicetopiperazinas , Eosinófilos/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Neutrófilos/efeitos dos fármacos , Ovalbumina/imunologia , Inibidores da Fosfodiesterase 4/farmacologia , Propionatos/farmacologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Quinolinas/farmacologia , Rolipram/farmacologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Fatores de TempoRESUMO
During the past 10 years, immunologists, epidemiologists and parasitologists have made many new exciting discoveries in the field of helminth-mediated immune regulation. In addition, many animal experiments have shown that certain helminths or products derived from helminths can protect mice from developing allergic or autoimmune disease. Some clinical trials utilising Trichuris suis or Necator americanus for the treatment of allergic disorders and inflammatory bowel disease have been conducted. The outcomes of these trials suggest that they may be used to treat these disorders. However, to date no helminth therapy is routinely being applied to patients and no helminth-derived product therapy has been developed. In order to bring new drugs to the market and shoulder the enormous costs involved in developing such therapies, pharmaceutical companies need to be involved. However, currently the resources from the pharmaceutical industry devoted to this concept are relatively small and there are good reasons why the industry may have been reluctant to invest in developing these types of therapies. In this review article, the hurdles that must be overcome before the pharmaceutical industry might invest in these novel therapies are outlined.
Assuntos
Indústria Farmacêutica/economia , Tratamento Farmacológico/economia , Helmintos/imunologia , Terapia com Helmintos/economia , Animais , Helmintos/química , Humanos , CamundongosRESUMO
The spleen tyrosine kinase (Syk) is a key mediator of immunoreceptor signaling in immune cells. Thus, interfering with the function of Syk by genetic deletion or pharmacological inhibition might influence a variety of allergic and autoimmune processes. Since conventional Syk knockout mice are not viable, studies addressing the effect of Syk deletion in adult animals have been limited. To further explore functions of Syk in animal models of allergy and to shed light on the role of Syk in the in vivo migration of neutrophils and monocytes, we generated inducible Syk knockout mice. These mice harbor a floxed Syk gene and a tamoxifen-inducible Cre recombinase under the control of the ubiquitously active Rosa26-promoter. Thus, treatment of mice with tamoxifen leads to the deletion of Syk in all organs. Syk-deleted mice were analyzed in mast cell-dependent models and in models focusing on neutrophil and monocyte migration. We show that Syk deletion in adult mice reduces inflammatory responses in mast cell-driven animal models of allergy and asthma but has no effect on the migration of neutrophils and monocytes. Therefore, the inducible Syk knockout mice presented here provide a valuable tool to further explore the role of Syk in disease-related animal models.
Assuntos
Movimento Celular , Quimiotaxia de Leucócito/imunologia , Hipersensibilidade/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Proteínas Tirosina Quinases/imunologia , Animais , Diferenciação Celular/imunologia , Separação Celular , Citometria de Fluxo , Inflamação/imunologia , Masculino , Mastócitos/citologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Neutrófilos/citologia , Transdução de Sinais/imunologia , Quinase SykRESUMO
Atopic asthma is a chronic inflammatory pulmonary disease characterised by recurrent episodes of wheezy, laboured breathing with an underlying Th2 cell-mediated inflammatory response in the airways. It is currently treated and, more or less, controlled depending on severity, with bronchodilators e.g. long-acting beta agonists and long-acting muscarinic antagonists or anti-inflammatory drugs such as corticosteroids (inhaled or oral), leukotriene modifiers, theophyline and anti-IgE therapy. Unfortunately, none of these treatments are curative and some asthmatic patients do not respond to intense anti-inflammatory therapies. Additionally, the use of long-term oral steroids has many undesired side effects. For this reason, novel and more effective drugs are needed. In this review, we focus on the CD4+ Th2 cells and their products as targets for the development of new drugs to add to the current armamentarium as adjuncts or as potential stand-alone treatments for allergic asthma. We argue that in early disease, the reduction or elimination of allergen-specific Th2 cells will reduce the consequences of repeated allergic inflammatory responses such as lung remodelling without causing generalised immunosuppression.
Assuntos
Asma/imunologia , Asma/terapia , Hipersensibilidade/terapia , Células Th2/imunologia , Corticosteroides/uso terapêutico , Agonistas Adrenérgicos beta/uso terapêutico , Animais , Antiasmáticos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Asma/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Terapia Genética/métodos , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Resultado do TratamentoRESUMO
Toll-like receptor (TLR) mediated signaling induces pro-inflammatory responses and can both suppress and exacerbate allergic responses in the airways. The aim of our study was to directly compare the efficacy of different TLR agonists in inhibiting or exacerbating the development of Th2-mediated responses in the airways and investigate if the suppressive effects were associated with increased pro-inflammatory responses. Mice were immunized on day 0, 14 and 21 by intraperitoneal injection of ovalbumin/alum and exposed to ovalbumin aerosol on day 26 and 27. TLR2, TLR3, TLR4, TLR7 and TLR9 agonists (0.001, 0.01, 0.1, or 1 mg/kg) were administered intratracheally 1 h before each allergen exposure. Both the TLR7 and TLR9 agonists dose dependently reduced airway eosinophilia, while the TLR3 agonist only reduced airway eosinophilia at a dose of 1.0 mg/kg. The TLR2 and TLR4 agonists potentiated eosinophilia. All TLR agonists enhanced neutrophil numbers at doses as low as 0.01 mg/kg, in particular TLR2 and TLR4 agonists. TLR7 and TLR9 agonists also significantly reduced IL-4 and IL-5 levels and all TLR agonists, with the exception of TLR7, enhanced the amount IL-1ß, IL-6, and TNF-α detected in the whole lung lavage. Only application of TLR9 agonist induced detectable levels of IL-10 in the lung. Suppressive effects of the TLR agonists were not dependent upon IFN-γ and IL-10 or associated with increased numbers of Foxp3(+)CD4(+) Tr cells in the lavage fluid. Airway resistance was reduced significantly only when TLR7 agonist was administered. When applied therapeutically 2 days after allergen exposure, all TLR agonists, except TLR2, similarly reduced airway eosinophilia and IL-4 levels. Taken together our results show that TLR7 agonists had the strongest anti-asthmatic effects with the lowest pro-inflammatory potential, suggesting that activating TLR7 may have the greatest potential to treat allergic disorders in humans.
Assuntos
Inflamação/etiologia , Interleucina-10/genética , Receptores Toll-Like/agonistas , Resistência das Vias Respiratórias/imunologia , Animais , Asma/tratamento farmacológico , Asma/imunologia , Relação Dose-Resposta a Droga , Eosinofilia/imunologia , Feminino , Inflamação/imunologia , Glicoproteínas de Membrana/agonistas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Ovalbumina/imunologia , Fatores de Tempo , Receptor 7 Toll-Like/agonistasRESUMO
BACKGROUND: Recent clinical trials, epidemiological studies and animal experiments have suggested that probiotics may help suppress the development of allergic responses. OBJECTIVE: To investigate whether the application of the probiotic Escherichia coli strain Nissle 1917 (EcN) protects mice from developing ovalbumin (OVA)-specific T helper-2 responses in the airways. METHODS: OVA-specific Th2 responses were induced by 2 intraperitoneal (i.p.) injections with OVA/alum followed by 1 intranasal (i.n.) challenge with OVA. EcN was given orally during the entire sensitization and challenge period, together with OVA/alum during the i.p. sensitizations, or i.n. before or during the airway challenge with OVA. RESULTS: We found that when the bacteria were given together with OVA/alum airway eosinophilia, airway hyper-reactivity, goblet cell metaplasia and IL-5 levels in the bronchoalveolar lavage and mediastinal lymph node cell cultures were reduced. This effect was associated with increased numbers of IFN-gamma producing T helper-1 cells and IFN-gamma levels in the airways and strongly increased OVA-specific IgG(2a) titers in the serum. The suppressive effect on airway eosinophilia was dependent on IFN-gamma but not TLR-4. Applying EcN i.n. or orally did not reduce the development of allergen-specific Th2 responses. CONCLUSIONS: Our results suggest that EcN can inhibit the development of allergic responses when the bacteria are present at the site of Th2 cell priming and that this immunomodulatory effect is due to a shift from Th2 to Th1 response. The data support the hypothesis that probiotics may help reduce allergic responses and that EcN may also be used as adjuvant therapy to induce allergen-specific Th1 responses.
Assuntos
Hiper-Reatividade Brônquica/prevenção & controle , Células Dendríticas/imunologia , Escherichia coli/imunologia , Hipersensibilidade/imunologia , Probióticos/uso terapêutico , Células Th2/imunologia , Adjuvantes Imunológicos/farmacologia , Administração Intranasal , Administração Oral , Alérgenos/imunologia , Compostos de Alúmen/farmacologia , Animais , Hiper-Reatividade Brônquica/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Eosinofilia/imunologia , Eosinofilia/metabolismo , Eosinofilia/microbiologia , Feminino , Células Caliciformes/imunologia , Células Caliciformes/patologia , Hipersensibilidade/metabolismo , Hipersensibilidade/microbiologia , Hipersensibilidade/prevenção & controle , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-5/biossíntese , Interleucina-5/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/microbiologia , Células Th2/metabolismo , Células Th2/microbiologiaRESUMO
Recent epidemiological and experimental data indicate that infection with helminths can protect humans from the development of allergic disorders by immunosuppressive mechanisms that involve the induction of IL-10 and/or regulatory T cells. Furthermore, helminth-derived immune modulators suppress allergic responses in mice. Trichuris suis therapy has been shown to be safe and efficacious in treating inflammatory bowel disease in humans. Has the time come to treat patients who have allergic diseases or healthy humans who are at risk of developing these diseases with helminths or helminth-derived products? Here, I discuss the pros and cons of such an approach.
Assuntos
Helmintíase/imunologia , Helmintos/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Animais , Antígenos de Helmintos/efeitos adversos , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/uso terapêutico , Extratos Celulares/imunologia , Extratos Celulares/uso terapêutico , Reações Cruzadas , Proteínas de Helminto/efeitos adversos , Proteínas de Helminto/imunologia , Proteínas de Helminto/uso terapêutico , Humanos , Hipersensibilidade/parasitologia , Tolerância Imunológica , Fatores Imunológicos/uso terapêutico , Interleucina-10/imunologia , Camundongos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/parasitologia , Células Th2/imunologia , Células Th2/parasitologiaRESUMO
Eosinophils play a major role in the development and severity of asthma. Robust and rapid preclinical animal models are desirable to profile novel therapeutics inhibiting the influx of eosinophils into the airways. To develop a rapid, airway eosinophil recruitment model in the rat, Brown-Norway (BN) rats were immunised with ovalbumin (OVA)/alum on day 0, 1 and 2 and challenged with OVA aerosol on day 5 and 6. On day 7 bronchoalveolar lavage fluid (BALF) was analysed for eosinophil numbers, eosinophil peroxidase (EPO) activity and cytokines. Lung sections were also examined. The immunised animals showed a strong selective influx of eosinophils into the airways correlating with enhanced EPO activity, Interleukin (IL-4), IL-5 and monocytes chemo attractant protein levels in the BALF in comparison to sham-sensitised rats. In addition the immunised rats developed goblet cell metaplasia in the lung and showed OVA specific IgG1 and IgE levels in the serum but no airway hyperreactivity after metacholine challenge. Airway inflammation was suppressed by applying the steroids Budesonide (intra tracheally) and Prednisolone (per orally), Roflumilast a phosphodiesterase-4 inhibitor, and the H1 receptor antagonists Epinastine and Ketotifen. Montelukast, a Leukotriene receptor antagonist and Chromoglycate, a mast cell stabiliser, had no effect in this model. In summary, in this novel preclinical rat model therapeutics expected to inhibit the development of airway eosinophilia can rapidly be tested.
Assuntos
Anti-Inflamatórios/farmacologia , Asma/fisiopatologia , Modelos Animais de Doenças , Eosinófilos/metabolismo , Compostos de Alúmen , Aminopiridinas/farmacologia , Animais , Asma/tratamento farmacológico , Benzamidas/farmacologia , Líquido da Lavagem Broncoalveolar , Budesonida/farmacologia , Ciclopropanos/farmacologia , Dibenzazepinas/farmacologia , Antagonistas dos Receptores Histamínicos H1 , Imidazóis/farmacologia , Cetotifeno/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina , Inibidores de Fosfodiesterase/farmacologia , Prednisolona/farmacologia , Ratos , Ratos Endogâmicos BNRESUMO
Th2 responses induced by allergens or helminths share many common features. However, allergen-specific IgE can almost always be detected in atopic patients, whereas helminth-specific IgE is often not detectable and anaphylaxis often occurs in atopy but not helminth infections. This may be due to T regulatory responses induced by the helminths or the lack of helminth-specific IgE. Alternatively non-specific IgE induced by the helminths may protect from mast cell or basophil degranulation by saturating IgE binding sites. Both of these mechanisms have been implicated to be involved in helminth-induced protection from allergic responses. An article in the current issue of the European Journal of Immunology describes the generation of an anti-Nippostrongylus brasiliensis-specific IgE antibody which was used to identify a novel N. brasiliensis antigen (Nb-Ag1). The authors demonstrated that Nb-Ag1 specific IgE could only be detected for a short period of time during infection, and that these levels were sufficient to prime mast cells thereby leading to active cutaneous anaphylaxis after the application of Nb-Ag1. This is the first report clearly showing that a low level of helminth-specific IgE, transiently produced, is able to induce mast cell degranulation in the presence of large amounts of polyclonal IgE.
Assuntos
Helmintíase/imunologia , Helmintos/imunologia , Hipersensibilidade/microbiologia , Imunoglobulina E/imunologia , Animais , Anticorpos Bloqueadores , Antígenos de Helmintos/imunologia , Humanos , Mastócitos/imunologiaRESUMO
Eosinophils represent one of the main effector cell populations of allergic airway inflammation and allergic bronchial asthma. Their infiltration correlates with many characteristics of the disease, including airway hyperresponsiveness (AHR) and increased mucus production. CCR-3 is the principle chemokine receptor involved in eosinophil attraction into inflamed tissue. Therefore, antagonizing CCR-3 could be a novel promising approach toward asthma therapy. We investigated the effect of a low-molecular-weight CCR-3 antagonist on established airway inflammation in a chronic model of experimental bronchial asthma. For this purpose, BALB/c mice intraperitoneally sensitized with ovalbumin (OVA) were chronically challenged with OVA aerosol to induce chronic airway inflammation and airway remodeling. The effect of antagonizing CCR-3 on asthma pathology was examined in BAL and lung histology. Airway reactivity was assessed by head-out body plethysmography. Treatment with the CCR-3 antagonist resulted in a marked reduction of eosinophils in the bronchoalveolar lumen and in airway wall tissue, whereas infiltration of lymphocytes or macrophages remained unchanged. The reduction in eosinophil infiltration was accompanied by normalization of AHR and prevention of goblet cell hyperplasia, indicating reduced mucus production. Furthermore, antagonizing CCR-3 prevented airway remodeling as defined by subepithelial fibrosis and increased accumulation of myofibrocytes in the airway wall of chronically challenged mice. These data demonstrate that antagonism of CCR3 reduces eosinophil numbers, which is accompanied by diminution of asthma pathology in a mouse model of established chronic experimental asthma. Therefore, antagonizing CCR-3 represents a new approach toward a promising asthma therapy.
Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Receptores de Quimiocinas/antagonistas & inibidores , Animais , Asma/imunologia , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/imunologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Doença Crônica , Modelos Animais de Doenças , Eosinófilos/imunologia , Feminino , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Receptores CCR3RESUMO
We analysed the effects of murine polyomavirus-like particles (PLPs) on bone marrow-derived dendritic cells (BMDCs) and T cells in vitro. BMDCs activated with PLPs up-regulated CD40, CD80, CD86 and major histocompatibility complex (MHC) class II surface markers and produced proinflammatory cytokines. Chimeric PLPs [expressing the ovalbumin (OVA)-peptides OVA(257-264) or OVA(323-339)], but not wildtype PLPs, activated OVA-specific CD8 T cells and OVA-specific CD4 T cells, respectively, indicating both MHC class I and II presentation of the peptides by antigen-presenting cells. Our results suggest that PLPs may be used as vaccine adjuvants priming dendritic cells to induce potent T cell responses.
Assuntos
Células Dendríticas/imunologia , Ativação Linfocitária , Polyomavirus/imunologia , Linfócitos T/imunologia , Adjuvantes Imunológicos , Animais , Apresentação de Antígeno , Antígenos CD/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Camundongos Endogâmicos C57BL , OvalbuminaRESUMO
RATIONALE: Epidemiological studies suggest that infections with helminths protect from the development of asthma. Supporting this view is our published finding that infection with Nippostrongylus brasiliensis decreased ovalbumin-induced Th2 responses in the lung of mice. OBJECTIVES: To evaluate if N. brasiliensis excretory-secretory products also prevent the development of asthma. METHODS: Mice were immunized with ovalbumin/alum intraperitoneally in the absence or presence of helminthic products and then challenged intranasally with ovalbumin. Six days later, we analyzed if the mice developed Th2 responses in the lung. MAIN RESULTS: The application of the helminthic products together with ovalbumin/alum during the sensitization period totally inhibited the development of eosinophilia and goblet cell metaplasia in the airways and also strongly reduced the development of airway hyperreactivity. Allergen-specific IgG1 and IgE serum levels were also strongly reduced. These findings correlated with decreased levels of IL-4 and IL-5 in the airways in product-treated animals. The suppressive effects on the development of allergic responses were independent of the presence of Toll-like receptors 2 and 4, IFN-gamma, and most important, IL-10. Interestingly, suppression was still observed when the helminthic products were heated or treated with proteinase K. Paradoxically, we found that strong helminth product-specific Th2 responses were induced in parallel with the inhibition of ovalbumin-specific responses. CONCLUSION: Our results suggest that helminths suppress the development of asthma by secreting substances that modulate allergic responses without affecting the generation of helminth-specific Th2 immunity. The identification of these products may lead to the design of novel therapeutic intervention strategies for the treatment of asthma.