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INTRODUCTION: Early-life exposure to antibiotics (ABX) has been linked to increases in asthma severity and prevalence in both children and laboratory animals. We explored the immunologic mechanisms behind this association using a mouse model of house dust mite (HDM)-induced asthma and early-life ABX exposure. METHODS: Mice were exposed to three short courses of ABX following weaning and experimental asthma was thereafter induced. Airway cell counts and differentials; serum immunoglobulin E (IgE); pulmonary function; lung histopathology; pulmonary regulatory T cells (Tregs); and the fecal microbiome were characterized following ABX exposure and induction of experimental asthma. RESULTS: Asthma severity was increased in mice exposed to ABX, including: airway eosinophilia, airway hyper-reactivity, serum HDM-specific IgE, and lung histopathology. ABX treatment led to sharp reduction in fecal microbiome diversity, including the loss of pro-regulatory organisms such as Lachnospira. Pulmonary Tregs were reduced with ABX treatment, and this reduction was directly proportional to diminished microbiome diversity. CONCLUSION: Intermittent exposure to ABX early in life worsened the severity of experimental asthma and reduced pulmonary Tregs; the latter change correlated with decreased microbiome diversity. These data may suggest targets for immunologic or probiotic therapy to counteract the harmful effects of childhood ABX.
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Antibacterianos/efeitos adversos , Asma/epidemiologia , Asma/etiologia , Pyroglyphidae , Linfócitos T Reguladores/citologia , Remodelação das Vias Aéreas , Alérgenos/imunologia , Animais , Antibacterianos/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Fezes/microbiologia , Feminino , Imunoglobulina E/sangue , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microbiota , Prevalência , RNA Ribossômico 16S/genética , Testes de Função Respiratória , Células Th2/citologiaRESUMO
BACKGROUND: Allergic asthma is a major cause of worldwide morbidity and results from inadequate immune regulation in response to innocuous, environmental antigens. The need exists to understand the mechanisms that promote nonreactivity to human-relevant allergens such as house dust mite (HDM) in order to develop curative therapies for asthma. The aim of our study was to compare the effects of short-, intermediate- and long-term HDM administration in a murine asthma model and determine the ability of long-term HDM exposure to suppress allergic inflammation. METHODS: C57BL/6 mice were intranasally instilled with HDM for short-term (2 weeks), intermediate-term (5 weeks) and long-term (11 weeks) periods to induce allergic airway disease (AAD). The severity of AAD was compared across all stages of the model via both immunological and pulmonary parameters. RESULTS: Short- and intermediate-term HDM exposure stimulated the development of AAD that included eosinophilia in the bronchoalveolar lavage fluid (BALF), pronounced airway hyperreactivity (AHR) and evidence of lung inflammation. Long-term HDM exposure promoted the suppression of AAD, with a loss of BALF eosinophilia and AHR despite persistent mononuclear inflammation in the lungs. Suppression of AAD with long-term HDM exposure was associated with an increase in both Foxp3+ regulatory T cells and IL-10-positive alveolar macrophages at the site of inflammation. CONCLUSIONS: This model recapitulates the key features of human asthma and may facilitate investigation into the mechanisms that promote immunological tolerance against clinically relevant aeroallergens.
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Asma/imunologia , Tolerância Imunológica/imunologia , Pneumonia/imunologia , Pyroglyphidae/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/sangue , Citocinas/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The role of CD8(+) T cells in the pathogenesis of asthma remains controversial, as both pro- and anti-inflammatory functions have been suggested. This study was designed to examine the endogenous CD8(+) T cell response in a biphasic ovalbumin (OVA)-induced model of allergic airway disease (AAD) and its subsequent resolution with the development of local inhalational tolerance (LIT). We observed increases in OVA-specific CD8(+) T cell numbers in the local lung compartments (bronchoalveolar lavage, lung tissue, hilar lymph node) at AAD and LIT; systemic compartments (spleen, inguinal lymph node) displayed no such increases in CD8(+) T cell numbers. OVA-specific CD8(+) T cells appeared to exhibit plasticity both phenotypically and functionally. They possessed pro-inflammatory characteristics at AAD, with high phenotypic expression of CD11a and increased functional expression of granzyme B and interferon-γ. In contrast, at LIT they showed increased phenotypic expression of the inhibitory marker NKG2A and functionally did not produce granzyme B or interferon-γ. In addition, in a discontinuous model the OVA-specific CD8(+) T cells could be recalled on re-exposure to OVA, demonstrating memory. Finally, confocal microscopy results showed that OVA-specific CD8(+) T cells at AAD are associated with B cell aggregates in lung tissue. These B cell aggregates resembled tertiary ectopic lymphoid tissue and may thus provide a local environment for the salient cellular interactions that contribute to the development of LIT.
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Linfócitos T CD8-Positivos/imunologia , Hipersensibilidade Respiratória/imunologia , Administração por Inalação , Animais , Linfócitos B/imunologia , Brônquios/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Agregação Celular/imunologia , Feminino , Granzimas/metabolismo , Tolerância Imunológica , Memória Imunológica , Imunofenotipagem , Interferon gama/metabolismo , Pulmão/imunologia , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Subfamília C de Receptores Semelhantes a Lectina de Células NK/análise , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Although functional asplenia from infarctions may be a major contributor to increased infectious mortality in sickle-cell disease (SCD), this relationship has not been fully defined. We used the transgenic Berkeley SCD mouse to define blood and splenic immunophenotypic differences in this model compared with C57BL/6 and hemizygous controls. In the serum of SCD mice, we found increased IgG2a and suppressed IgM, IgG2b, and IgA levels. Serum IL-6 levels in SCD mice were elevated, whereas IL-1α, CXCL10, and CCL5 levels were decreased. The blood of SCD mice had higher white blood cell counts, with an increased percentage of lymphocytes and decreases in other leukocytes. Immunophenotyping of lymphocytes revealed higher percentages of CD8(+) and T-regulatory cells and lower percentages of B cells. SCD mouse spleens exhibited histological disorganization, with reduction of defined lymphoid follicles and expansion of red pulp, a greater than fourfold increase in splenic mononuclear cells, marked expansion of the nucleated red blood cell fraction, and B-cell and CD8(+) T-cell lymphopenia. Within the splenic B-cell population, there was a significant decrease in B-1a B cells, with a corresponding decrease in IgA secreting plasma cells in the gut. Confocal microscopy of spleens demonstrated complete disruption of the normal lymphofollicular structure in the white pulp of SCD mice without distinct B, T, and marginal zones. Our findings suggest that altered SCD splenic morphological characteristics result in an impaired systemic immune response.
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Anemia Falciforme/imunologia , Anemia Falciforme/patologia , Imunidade/imunologia , Baço/imunologia , Baço/patologia , Anemia Falciforme/sangue , Anemia Falciforme/complicações , Animais , Linfócitos B/imunologia , Quimiocinas/sangue , Modelos Animais de Doenças , Feminino , Hemizigoto , Imunoglobulinas/sangue , Interleucina-1alfa/sangue , Interleucina-6/sangue , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Soro , Linfócitos T/imunologiaRESUMO
BACKGROUND: Children with sickle cell disease (SCD) are susceptible to recurrent infections, which are often life threatening and necessitate frequent vaccinations. Given the altered baseline immunity and proinflammatory state associated with SCD, we sought to determine the relative safety and efficacy of vaccination in transgenic SCD mice. METHODS: Eight-week-old SCD mice were vaccinated with ovalbumin and aluminum hydroxide weekly for 3 wk by the intraperitoneal or intramuscular route. One week after the third vaccination, serum cytokines/chemokines, immunoglobulins, and bronchoalveolar lavage fluid cytokines were measured. RESULTS: Only SCD mice were prone to mortality associated with vaccination, as 40% of the animals died after the intraperitoneal vaccinations and 50% died after the intramuscular vaccinations. Serum IgG2b and IgM were significantly lower in SCD mice than in C57BL/6 mice after vaccination, but ovalbumin-specific IgE was significantly higher. Serum interleukin (IL)-1α, IL-2, IL-5, macrophage inflammatory protein 1α, and granulocyte macrophage-colony stimulating factor were significantly lower in SCD mice than in C57BL/6 mice after vaccination, whereas bronchoalveolar lavage fluid IL-1ß and IL-6 were increased. CONCLUSION: Mice with SCD appear to have a dysregulated immune response to vaccination. Thus, the relative safety and immunogenicity of vaccination should be studied in greater detail in the context of SCD.
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Anemia Falciforme/imunologia , Vacinação/efeitos adversos , Vacinação/mortalidade , Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/efeitos adversos , Análise de Variância , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/sangue , Citocinas/imunologia , Feminino , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Injeções Intramusculares , Camundongos , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/efeitos adversosRESUMO
Bromelain (Br) is a cysteine peptidase (GenBank AEH26024.1) from pineapple, with over 40 years of clinical use. The constituents mediating its anti-inflammatory activity are not thoroughly characterized and no peptide biomarker exists. Our objective is to characterize Br raw material and identify peptides in the plasma of Br treated mice. After SDS-PAGE in-gel digestion, Br (VN#3507; Middletown, CT, USA) peptides were analyzed via LC/MS/MS using 95% protein probability, 95% peptide probability, and a minimum peptide number = 5. Br spiked mouse plasma (1 ug/ul) and plasma from i.p. treated mice (12 mg/kg) were assessed using SRM. In Br raw material, we identified seven proteins: four proteases, one jacalin-like lectin, and two protease inhibitors. In Br spiked mouse plasma, six proteins (ananain, bromelain inhibitor, cysteine proteinase AN11, FB1035 precursor, FBSB precursor, and jacalin-like lectin) were identified. Using LC/MS/MS, we identified the unique peptide, DYGAVNEVK, derived from FB1035, in the plasma of i.p. Br treated mice. The spectral count of this peptide peaked at 6 hrs and was undetectable by 24 hrs. In this study, a novel Br peptide was identified in the plasma of treated mice for the first time. This Br peptide could serve as a biomarker to standardize the therapeutic dose and maximize clinical utility.
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CONTEXT: Allergic asthma continues to increase despite new pharmacological advances for both acute treatment and chronic-disease management. Asthma is a multifactorial disease process with genetic, allergic, infectious, environmental, and dietary origins. Researchers are investigating the benefits of lifestyle changes and alternative asthma treatments, including the ability of bromelain to inhibit inflammation. Bromelain is a commonly used, proteolytically active pineapple extract. OBJECTIVE: The present study intended to determine the ability of bromelain to reduce the inflammation of preexisting asthma via an ovalbumin (OVA)-induced murine model of allergic airway disease (AAD). DESIGN: The research team designed a study examining the effects of bromelain in a control group of mice that received phosphate buffered saline (PBS) only and in an intervention group that received bromelain in PBS. Setting The study took place in the Department of Immunology at the University of Connecticut's School of Medicine, Farmington. Intervention The research team sensitized female C57BL/6J mice with intraperitoneal OVA/alum and then challenged them with OVA aerosolization for 10 consecutive days. On day 4, the team began administering daily doses of PBS to the control group (n = 10) and bromelain (6mg/kg) in PBS to the bromelain (intervention) group (n = 10). OUTCOME MEASURES: The primary measures included bronchoalveolar lavage (BAL) cellular differential, cellular phenotype via flow cytometry, and lung histology. Additional outcomes included testing for serum cytokines and immunoglobulin. RESULTS: Bromelain treatment of AAD mice (bromelain group) resulted in significant anti-inflammatory activity as indicated by reduced BAL total leukocytes (P < .05), eosinophils (P < .05), and cellular infiltrates via lung pathology (P < .005), as compared to the control group. In addition, bromelain significantly reduced BAL CD4+ and CD8+ T cells without affecting cell numbers in the spleen or hilar lymph node. The study found decreased interleukins IL-4, IL-12, IL-17, as well as IFN-α in the serum of bromelain-treated animals. CONCLUSIONS: The results suggest that bromelain has a therapeutic effect in established AAD, which may translate into an effective adjunctive therapy in patients with similar conditions, such as allergic asthma, who have chosen to initiate treatment after the onset of symptoms.
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Anti-Inflamatórios não Esteroides/farmacologia , Asma/tratamento farmacológico , Asma/imunologia , Bromelaínas/farmacologia , Alérgenos/imunologia , Animais , Asma/prevenção & controle , Líquido da Lavagem Broncoalveolar/imunologia , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina , Receptores de Fator de Crescimento Neural/imunologia , Receptores do Fator de Necrose Tumoral/imunologiaRESUMO
Staphylococcus aureus, a primary source of bacterial superantigen (SAg), is known to colonize the human respiratory tract and has been implicated in airway inflammation. Studies have documented a role for SAgs in respiratory disorders, such as nasal polyps, chronic obstructive pulmonary disease, chronic rhinosinusitis, and asthma. However, cellular and molecular mediators involved in SAg-mediated pulmonary disease have not been clearly identified. In this study, we investigated the effect of intranasal staphylococcal enterotoxin A (SEA) exposure on murine lung. The pathological features in the lung resulting from SEA exposure had characteristics of both obstructive and restrictive pulmonary disorders. There was also an increase in bronchoalveolar lavage protein concentration and cellularity following SEA challenge. Massive CD8(+)Vbeta3(+) T cell accumulation observed in the lung was dependent on CD4 T cell help, both for recruitment and for IFN-gamma synthesis. The primary source of IFN-gamma synthesis was CD8 T cells, and depletion of these cells abrogated disease. IFN-gamma deficiency also prevented SEA-mediated disease, and this was by enhancing early recruitment of neutrophils as detected in the bronchoalveolar lavage. Thus, IFN-gamma appeared to selectively aid the recruitment of T cells to the lungs while preventing the neutrophil accumulation. Therefore, our results show that IFN-gamma-producing CD8 T cells mediated pulmonary alveolitis and inflammation, which were dependent upon CD4 T cells for their recruitment to the lung.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Enterotoxinas/farmacologia , Interferon gama/biossíntese , Pneumopatias/etiologia , Animais , Linfócitos T CD4-Positivos , Quimiotaxia de Leucócito , Enterotoxinas/administração & dosagem , Inalação , Pulmão , Camundongos , NeutrófilosRESUMO
BACKGROUND: Mice sensitized to ovalbumin develop allergic airway disease (AAD) with short-term aerosol challenge; however, airway inflammation resolves with long-term aerosol challenge, referred to as local inhalational tolerance (LIT). METHODS: We sought to determine if resolution of airway inflammation correlated with increases in lymphocyte subsets in local lung compartments, including putative regulatory T cells. RESULTS: At the AAD stage, total numbers of T and B lymphocytes in bronchoalveolar lavage (BAL) were significantly increased above controls; however, at LIT, T and B lymphocytes were significantly reduced compared to AAD. In the lung tissue, the only alteration was a significant increase in CD4+ CD25+ T cells at AAD. In the hilar lymph node (HLN), CD4+ and CD4+ CD25+ T cells were significantly increased at AAD and LIT. In addition, CD8+ T cells were significantly elevated in the HLN at LIT, and CD19+ B cells were significantly increased at AAD. Adoptive transfer of HLN lymphocytes to lymphopenic mice confirmed that AAD lymphocytes could induce airway inflammation in response to aerosol challenge, whereas LIT lymphocytes were unable to do so. Depletion of CD4+ CD25+ T cells in vivo resulted in exacerbation of inflammation at AAD and LIT. CD4+ CD25+ T cells in the HLN also displayed suppressive activity in vitro. Additionally, T cells expressing Foxp3 were increased in the BAL and HLN during LIT. CONCLUSIONS: These results indicate that lymphocytes with regulatory functions are increased and sustained in local lung compartments at LIT and that their appearance correlates with the resolution of lung inflammation.
Assuntos
Asma/imunologia , Tolerância Imunológica , Pulmão/imunologia , Linfonodos/imunologia , Hipersensibilidade Respiratória/imunologia , Linfócitos T Reguladores/imunologia , Aerossóis , Alérgenos/imunologia , Animais , Antígenos CD19/metabolismo , Asma/patologia , Linfócitos B/citologia , Linfócitos B/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Antígenos CD4/metabolismo , Contagem de Células , Proliferação de Células , Doença Crônica , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Inflamação/patologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Hipersensibilidade Respiratória/patologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismoRESUMO
Abolishing the inhibitory signal of intracellular cAMP is a prerequisite for effector T (Teff) cell function. The regulation of cAMP within leukocytes critically depends on its degradation by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE8A, a PDE isoform with 40-100-fold greater affinity for cAMP than PDE4, is selectively expressed in Teff vs. regulatory T (Treg) cells and controls CD4(+) Teff cell adhesion and chemotaxis. Here, we determined PDE8A expression and function in CD4(+) Teff cell populations in vivo. Using magnetic bead separation to purify leukocyte populations from the lung draining hilar lymph node (HLN) in a mouse model of ovalbumin-induced allergic airway disease (AAD), we found by Western immunoblot and quantitative (q)RT-PCR that PDE8A protein and gene expression are enhanced in the CD4(+) T cell fraction over the course of the acute inflammatory disease and recede at the late tolerant non-inflammatory stage. To evaluate PDE8A as a potential drug target, we compared the selective and combined effects of the recently characterized highly potent PDE8-selective inhibitor PF-04957325 with the PDE4-selective inhibitor piclamilast (PICL). As previously shown, PF-04957325 suppresses T cell adhesion to endothelial cells. In contrast, we found that PICL alone increased firm T cell adhesion to endothelial cells by ~20% and significantly abrogated the inhibitory effect of PF-04957325 on T cell adhesion by over 50% when cells were co-exposed to PICL and PF-04957325. Despite its robust effect on T cell adhesion, PF-04957325 was over two orders of magnitude less efficient than PICL in suppressing polyclonal Teff cell proliferation, and showed no effect on cytokine gene expression in these cells. More importantly, PDE8 inhibition did not suppress proliferation and cytokine production of myelin-antigen reactive proinflammatory Teff cells in vivo and in vitro. Thus, targeting PDE8 through PF-04957325 selectively regulates Teff cell interactions with endothelial cells without marked immunosuppression of proliferation, while PDE4 inhibition has partially opposing effects. Collectively, our data identify PF-04957325 as a novel function-specific tool for the suppression of Teff cell adhesion and indicate that PDE4 and PDE8 play unique and non-redundant roles in the control of Teff cell functions.
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Mice sensitized to ovalbumin (OVA) develop allergic airway disease (AAD) with short-term daily OVA aerosol challenge; inflammation resolves with long-term OVA aerosol exposure, resulting in local inhalational tolerance (LIT). Cbl-b is an E3 ubiquitin ligase involved with CD28 signaling; Cbl-b(-/-) effector T cells are resistant to regulatory T cell-mediated suppression in vitro and in vivo. The present study utilized Cbl-b(-/-) mice to investigate the role of Cbl-b in the development of AAD and LIT. Cbl-b(-/-) mice exhibited increased airway inflammation during AAD, which failed to resolve with long-term OVA aerosol exposure. Exacerbation of inflammation in Cbl-b(-/-) mice correlated with increased proinflammatory cytokine levels and expansion of effector T cells in the BAL during AAD, but did not result in either a modulation of lymphocyte subsets in systemic tissues or in OVA-specific IgE in serum. These results implicate a role for Cbl-b in the resolution of allergic airway inflammation.
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Comorbid asthma in sickle cell disease (SCD) confers higher rates of vaso-occlusive pain and mortality, yet the physiological link between these two distinct diseases remains puzzling. We used a mouse model of SCD to study pulmonary immunology and physiology before and after the induction of allergic airway disease (AAD). SCD mice were sensitized with ovalbumin (OVA) and aluminum hydroxide by the intraperitoneal route followed by daily, nose-only OVA-aerosol challenge to induce AAD. The lungs of naive SCD mice showed signs of inflammatory and immune processes: (1) histologic and cytochemical evidence of airway inflammation compared with naive wild-type mice; (2) bronchoalveolar lavage (BAL) fluid contained increased total lymphocytes, %CD8+ T cells, granulocyte-colony stimulating factor, interleukin 5 (IL-5), IL-7, and chemokine (C-X-C motif) ligand (CXCL)1; and (3) lung tissue and hilar lymph node (HLN) had increased CD4+, CD8+, and regulatory T (Treg) cells. Furthermore, SCD mice at AAD demonstrated significant changes compared with the naive state: (1) BAL fluid with increased %CD4+ T cells and Treg cells, lower %CD8+ T cells, and decreased interferon gamma, CXCL10, chemokine (C-C motif) ligand 2, and IL-17; (2) serum with increased OVA-specific immunoglobulin E, IL-6, and IL-13, and decreased IL-1α and CXCL10; (3) no increase in Treg cells in the lung tissue or HLN; and (4) hyporesponsiveness to methacholine challenge. In conclusion, SCD mice have an altered immunologic pulmonary milieu and physiological responsiveness. These findings suggest that the clinical phenotype of AAD in SCD mice differs from that of wild-type mice and that individuals with SCD may also have a unique, divergent phenotype perhaps amenable to a different therapeutic approach.
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Anemia Falciforme/complicações , Anemia Falciforme/imunologia , Hipersensibilidade/complicações , Hipersensibilidade/imunologia , Inflamação/complicações , Inflamação/imunologia , Pulmão/patologia , Anemia Falciforme/sangue , Animais , Asma/sangue , Asma/complicações , Asma/imunologia , Hiper-Reatividade Brônquica/sangue , Hiper-Reatividade Brônquica/complicações , Hiper-Reatividade Brônquica/imunologia , Líquido da Lavagem Broncoalveolar , Citocinas/sangue , Modelos Animais de Doenças , Eosinófilos/metabolismo , Feminino , Hemizigoto , Hipersensibilidade/sangue , Inflamação/sangue , Contagem de Leucócitos , Pulmão/imunologia , Camundongos Endogâmicos C57BL , Muco/metabolismo , Ovalbumina/imunologia , Linfócitos T/imunologiaRESUMO
B cells have long been known to participate in both innate and adaptive immune responses by contributing to antigen presentation and by producing antigen-specific antibodies. Recent evidence shows that certain B-cell subsets can also inhibit T-cell immune responses. Like regulatory T cells (Treg), these regulatory B cells (Breg) appear to comprise several subpopulations. How Breg cells are generated and how they control immune responses in vivo are just beginning to be elucidated. Here, we provide detailed instructions for the identification, isolation, and functional characterization of Breg cells in a murine model of allergic airway disease.
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Linfócitos B Reguladores/imunologia , Linfócitos B Reguladores/patologia , Modelos Animais de Doenças , Camundongos/imunologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Animais , Lavagem Broncoalveolar/métodos , Separação Celular/métodos , Crioultramicrotomia/métodos , Microscopia Confocal/métodos , Hipersensibilidade Respiratória/sangue , Coloração e Rotulagem/métodos , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The incidence of atopic conditions has increased in industrialized countries. Persisting symptoms and concern for drug side-effects lead patients toward adjunctive treatments such as phytotherapy. Previously, we have shown that Bromelain (sBr), a mixture of cysteine proteases from pineapple, Ananas comosus, inhibits ovalbumin (OVA)-induced murine model of allergic airway disease (AAD). However, sBr's effect on development of AAD when treatment is administered throughout OVA-alum sensitization was unknown and is the aim of the present study. C57BL/6J mice were sensitized with OVA/alum and challenged with 7 days OVA aerosol. sBr 6 mg/kg/0.5 ml or PBS vehicle were administered throughout sensitization. Lung, bronchoalveolar lavage (BAL), spleen, and lymph nodes were processed for flow cytometry and OVA-specific IgE was determined via ELISA. sBr treatment throughout OVA-alum sensitization significantly reduced the development of AAD (BAL eosinophils and lymphocytes). OVA-specific IgE and OVA TET(+) cells were decreased. sBr reduced CD11c(+) dendritic cell subsets, and in vitro treatment of DCs significantly reduced CD44, a key receptor in both cell trafficking and activation. sBr was shown to reduce allergic sensitization and the generation of AAD upon antigen challenge. These results provide additional insight into sBr's anti-inflammatory and antiallergic properties and rationale for translation into the clinical arena.
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Bromelain (Br), an extract from pineapple stem with cysteine protease activity, exerts anti-inflammatory effects in a number of inflammatory models. We have previously shown that Br treatment decreased activated CD4(+) T cells and has a therapeutic role in an ovalbumin-induced murine model of allergic airway disease. The current study was designed to determine the effect of Br on CD4(+) T cell activation, specifically the expression of CD25 in vitro. CD25 is up regulated upon T cell activation, found as a soluble fraction (sCD25) and is a therapeutic target in inflammation, autoimmunity and allergy. Br treatment of anti-CD3 stimulated CD4(+) T cells reduced CD25 expression in a dose and time dependent manner. This reduction of CD25 was dependent on the proteolytic action of Br as the addition of E64 (a cysteine protease inhibitor) abrogated this response. The concentration of sCD25 was increased in supernatants of Br treated activated CD4(+) T cells as compared to control cells, suggesting that Br proteolytically cleaved cell-surface CD25. This novel mechanism of action identifies how Br may exert its therapeutic benefits in inflammatory conditions.
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Anti-Inflamatórios não Esteroides/farmacologia , Bromelaínas/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Ananas/química , Animais , Bromelaínas/química , Linfócitos T CD4-Positivos/imunologia , Extratos Celulares/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Subunidade alfa de Receptor de Interleucina-2/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Bromelain, a widely used pineapple extract with cysteine protease activity, has been shown to have immunomodulatory effects in a variety of immune system models. The purpose of the present study was to determine the effects of orally administered bromelain in an ovalbumin (OVA)-induced murine model of acute allergic airway disease (AAD). To establish AAD, female C57BL/6J mice were sensitized with intraperitoneal (i.p.) OVA/alum and then challenged with OVA aerosols for 3 days. Mice were gavaged with either (phosphate buffered saline)PBS or 200 mg/kg bromelain in PBS, twice daily for four consecutive days, beginning 1 day prior to OVA aerosol challenge. Airway reactivity and methacholine sensitivity, bronchoalveolar lavage (BAL) cellular differential, Th2 cytokines IL-5 and IL-13, and lung histology were compared between treatment groups. Oral bromelain-treatment of AAD mice demonstrated therapeutic efficacy as evidenced by decreased methacholine sensitivity (P = 0.01), reduction in BAL eosinophils (P = 0.02) and IL-13 concentrations (P = 0.04) as compared with PBS controls. In addition, oral bromelain significantly reduced BAL CD19+ B cells (P = 0.0001) and CD8+ T cells (P = 0.0001) in AAD mice when compared with controls. These results suggest that oral treatment with bromelain had a beneficial therapeutic effect in this murine model of asthma and bromelain may also be effective in human conditions.
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Acute exposure of sensitized mice to antigen elicits allergic airway disease (AAD) characterized by Th2 cytokine-dependent pulmonary eosinophilia, methacholine hyperresponsiveness and antigen-specific IgE elevation. However, chronic exposure induces a local inhalational tolerance (LIT), with resolution of the airway responses but persistent systemic IgE production. To further determine if systemic immunologic responses were maintained during LIT, we assessed subcutaneous late phase responses to ovalbumin in this model. Sensitized and AAD mice developed small subcutaneous responses to ovalbumin, with footpad thickness increasing to 113.7 and 113.6% of baseline, respectively. In comparison, LIT mice developed marked foot swelling (141.6%). Histologic examination confirmed increased inflammation in the chronic animals, with a significant contribution by eosinophils. Thus, the resolution of airway inflammatory responses with chronic antigen inhalation is a localized response, not associated with loss of systemic responses to antigen.
Assuntos
Asma/imunologia , Hiper-Reatividade Brônquica/imunologia , Modelos Animais de Doenças , Edema/etiologia , Eosinófilos/imunologia , Tolerância Imunológica/fisiologia , Administração por Inalação , Animais , Asma/induzido quimicamente , Asma/patologia , Eosinófilos/patologia , Feminino , Pé/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/administração & dosagem , Ovalbumina/farmacologiaRESUMO
Mice sensitized to OVA and subjected to acute OVA aerosol exposures develop allergic airway disease (AAD). However, chronic continuous Ag exposure results in resolution of AAD and the development of local inhalational tolerance (LIT). Because we have previously observed the persistence of B cells in the bronchoalveolar lavage (BAL) and hilar lymph nodes (HLN) at the resolution stage of this model, we investigated the role of B cells in the modulation of AAD. Although B cell-deficient mice developed LIT, adoptive transfer of HLN B cells from LIT mice to OVA-sensitized recipients resulted in attenuated AAD following subsequent OVA aerosol exposure, as determined by reduced BAL leukocytosis and eosinophilia, decreased tissue inflammation, and absent methacholine hyper-responsiveness. In similar adoptive transfer studies, HLN B cells from AAD mice were without effect. The protection transferred by LIT HLN B cells was Ag specific and was associated with accumulation of Foxp3(+) T regulatory cells regionally in BAL and HLN, but not systemically in the spleen. Fluorescent labeling of LIT HLN B cells before adoptive transfer demonstrated that these cells had the capacity to migrate to local inflammatory sites. In vitro assessment demonstrated that the LIT HLN B cells exerted this regulatory effect via TGF-beta induced conversion of CD4(+)CD25(-) T effector cells into functionally suppressive CD4(+)CD25(+)Foxp3(+) T regulatory cells. These findings illustrated a novel regulatory role for regional B cells in AAD and suggested a possible contributory role of B cells, along with other cell types, in the establishment of LIT.
Assuntos
Linfócitos B/imunologia , Hipersensibilidade Respiratória/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/metabolismo , Transferência Adotiva , Animais , Subpopulações de Linfócitos B/imunologia , Linfócitos B/metabolismo , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Tolerância Imunológica , Pulmão/citologia , Pulmão/imunologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Ovalbumina/imunologia , Hipersensibilidade Respiratória/metabolismo , Baço/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/imunologiaRESUMO
The expression of the mouse Cyp family and key inflammatory mediators were examined in a model of ovalbumin (OVA)-induced allergic airway disease. The expression of IL-4, IL-13 and Ccl11 increased during the acute phase of allergic inflammation and decreased with its resolution. Interestingly, the expression of Ccl20 was increased during the resolution phase. The response of the Cyp gene family to the development of allergic inflammation was differential and correlated with the evolution of the inflammatory response. During the acute inflammatory phase the mRNA levels of Cyp2e1, Cyp2f2, Cyp2j6, Cyp4b1, Cyp8a1 and Cypor were decreased while the mRNA levels of Cyp4f18, Cyp5a1 and Cyp7b1 were elevated. With resolution of the inflammation the expression patterns returned to normal. These changes suggest that the Cyp family may play a role in the allergic inflammation by modulating the metabolism of xenobiotics and endogenous compounds such as LTB4, TXA1, PGI2 and native anti-glucocorticoids.
Assuntos
Sistema Enzimático do Citocromo P-450/imunologia , Imunidade Inata/imunologia , Fatores Imunológicos/imunologia , Pulmão/imunologia , Pneumonia/imunologia , Animais , Citocromo P-450 CYP2J2 , Feminino , Pulmão/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/enzimologiaRESUMO
OBJECTIVE: IL-10 is a potent anti-inflammatory cytokine, and IL-10-producing regulatory T cells are effective inhibitors of murine asthmatic responses. This study determined whether IL-10-dependent mechanisms mediated the local inhalational tolerance seen with chronic inhalational exposure to antigen. METHODS: Wildtype and IL-10(-/-) mice were sensitized with ovalbumin (OVA) and then challenged with daily OVA inhalations for 10 days or 6 weeks. RESULTS: The 10-day animals developed allergic airway disease, characterized by BAL eosinophilia, histologic airway inflammation and mucus secretion, methacholine hyperresponsiveness, and OVA-specific IgE production. These changes were more pronounced in IL-10(-/-) mice. The 6-week IL-10(-/-) and wildtype animals both developed inhalational tolerance, with resolution of airway inflammation but persistence of OVA-specific IgE production. CONCLUSION: IL-10 may have anti-inflammatory effects in the acute stage of murine allergic airways disease, but the cytokine does not mediate the development of local inhalational tolerance with chronic antigen exposure.