RESUMO
Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.
Assuntos
Pulmão/imunologia , Mucina-5B/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Animais , Asma/imunologia , Asma/metabolismo , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Cílios/fisiologia , Orelha Média/imunologia , Orelha Média/microbiologia , Feminino , Inflamação/patologia , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Mucina-5AC/deficiência , Mucina-5AC/metabolismo , Mucina-5B/deficiência , Mucina-5B/genética , Fagocitose , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/microbiologia , Staphylococcus aureus/imunologia , Análise de SobrevidaRESUMO
Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.
Assuntos
Ativação do Complemento/fisiologia , Complemento C3/imunologia , Complemento C5a/imunologia , Animais , Complexo Antígeno-Anticorpo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Complemento C3/genética , Complemento C5a/genética , Humanos , Imunoglobulina G/imunologia , Fígado/citologia , Fígado/metabolismo , Pulmão/imunologia , Pulmão/patologia , Lesão Pulmonar , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Protrombina/genética , Protrombina/metabolismo , Trombina/metabolismoRESUMO
Complement regulatory proteins prevent excessive complement system activation and deposition, which can lead to host tissue damage, including fetal loss during pregnancy. To further understand the regulation of complement during development, we examined the expression of the complement protein, C3, and the active subunit of carboxypeptidase N (CPN1), the complement anaphylatoxin regulator. RNA and protein analyses indicated that CPN1 expression occurred as early as 8.5 days post coitus (dpc) and continued through birth. At 10.5 and 13.5 dpc, in situ hybridization revealed CPN1 RNA in erythroid progenitor cells. At 16.5 dpc, expression of CPN1 was also detected in hepatocytes. In comparison to CPN1, C3 RNA expression occurred later (after 13.5 dpc). Moreover, C3 expression was limited to the liver erythroid progenitor cells at 16.5 dpc. These results demonstrated that mouse embryos contain RNA and protein for both C3 and CPN1, and CPN1 expression precedes that of C3 by several days.
Assuntos
Complemento C3/biossíntese , Desenvolvimento Embrionário e Fetal/imunologia , Lisina Carboxipeptidase/biossíntese , Camundongos Endogâmicos C57BL/embriologia , Animais , Western Blotting/veterinária , Complemento C3/genética , Complemento C3/imunologia , Feminino , Proteínas Fetais/biossíntese , Proteínas Fetais/genética , Proteínas Fetais/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Fígado/embriologia , Fígado/imunologia , Lisina Carboxipeptidase/genética , Lisina Carboxipeptidase/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos C57BL/imunologia , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
Accumulating evidence demonstrates that both Th2 and Th17 responses are involved in the pathogenesis of allergic airway inflammation in animals as well as in humans. The lung contains diverse types of antigen presenting cells. However, the mechanism by which these antigen presenting cells regulate Th2 versus Th17 responses in the lung remains incompletely understood. Here, we show that intranasal administration of fungal protease allergen induced both Th2 and Th17 responses in the lung with different kinetics. Notably, depletion of CD11c(+) cells or macrophages greatly diminished the numbers of allergen-specific Th2 cells in the lung, the infiltration of eosinophils into the airway and airway hyperreactivity. In sharp contrast, depletion of the same antigen presenting cells significantly increased the numbers of allergen-specific Th17 cells in the lung and the infiltration of neutrophils into the airway. Moreover, although a subpopulation of lung epithelial cells express MHC II, lack of MHC II expression in parenchymal cells did not alter pulmonary Th2 and Th17 responses. Our results demonstrate that antigen presenting cells differentially regulate the generation of pulmonary Th2 and Th17 cells in response to intranasal protease allergens.
Assuntos
Alérgenos/imunologia , Células Apresentadoras de Antígenos/imunologia , Pulmão/imunologia , Células Th17/imunologia , Células Th2/imunologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Aspergillus/imunologia , Aspergillus/metabolismo , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Eosinófilos/imunologia , Eosinófilos/metabolismo , Proteínas Fúngicas/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Peptídeo Hidrolases/imunologia , Pneumonia/genética , Pneumonia/imunologia , Pneumonia/metabolismo , Células Th17/metabolismo , Células Th2/metabolismoRESUMO
Activation of complement is one of the earliest immune responses to exogenous threats, resulting in various cleavage products including anaphylatoxin C3a. In addition to its contribution to host defense, C3a has been shown to mediate Th2 responses in animal models of asthma. However, the role of C3a on pulmonary Th17 responses during allergic inflammation remains unclear. Here, we show that mice deficient in C3a receptor (C3aR) exhibited (i) higher percentages of endogenous IL-17-producing CD4(+) T cells in the lungs, (ii) higher amounts of IL-17 in the bronchoalveolar lavage fluid, and (iii) more neutrophils in the lungs than wild-type mice when challenged with intranasal allergens. Moreover, adoptive transfer experiments showed that the frequencies of antigen-specific IL-17-producing CD4(+) T cells were significantly higher in the lungs and bronchial lymph nodes of C3aR-deficient recipients than those of wild-types recipients. Bone-marrow reconstitution study indicated that C3aR-deficiency on hematopoietic cells was required for the increased Th17 responses. Furthermore, C3aR-deficient mice exhibited increased percentages of Foxp3(+) regulatory T cells; however, depletion of these cells minimally affected the induction of antigen-specific Th17 cell population in the lungs. Neutralization of IL-17 significantly reduced the number of neutrophils in bronchoalveolar lavage fluid of C3aR-deficient mice. Our findings demonstrate that C3a signals negatively regulate antigen-specific Th17 responses during allergic lung inflammation and the size of Foxp3(+) regulatory T cell population in the periphery.
Assuntos
Anafilatoxinas/imunologia , Complemento C3a/imunologia , Hipersensibilidade/imunologia , Pneumonia/imunologia , Células Th17/imunologia , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Complemento C3a/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Hipersensibilidade/metabolismo , Interleucina-17/biossíntese , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Pneumonia/genética , Receptores de Complemento/genética , Receptores de Complemento/imunologia , Receptores de Complemento/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
RATIONALE: The factors that control the secretion of epithelial mucins are essential to understanding obstructive airway diseases such as asthma. Although the complement anaphylatoxin C3a and its receptor have been shown to promote many features of allergic lung inflammation, the contribution to mucin expression has not been elucidated. OBJECTIVES: To determine if the C3a receptor with its ligand regulates airway epithelial mucin production. METHODS: Mice deficient in the C3a receptor were examined in a model of allergic airway disease for the presence of goblet cells and the gel-forming secreted mucin Muc5ac. MEASUREMENTS AND MAIN RESULTS: Lungs from antigen-challenged C3a receptor-deficient mice revealed a dramatic decrease in goblet cells and Muc5ac compared with challenged wild-type control animals. These differences were dependent on C3a binding to its receptor since intranasal challenge with C3a induced the formation of goblet cells only in wild-type but not C3a receptor-deficient mice. Increased numbers of goblet cells were also found in C3a-stimulated RAG-1-deficient mice demonstrating a mechanism independent of T lymphocytes and Th2 cytokines, mediators which have been shown to regulate mucin expression. A direct physiological role for C3a in these models was further demonstrated in cultures of airway epithelial Clara cells, which not only express the C3a receptor but also produce Muc5ac in response to C3a. CONCLUSIONS: These studies identify a novel C3a receptor-dependent mechanism in the development of airway epithelial goblet cells and regulation of Muc5ac production and implicate C3a as a mediator of airway obstruction in asthma.
Assuntos
Asma/fisiopatologia , Complemento C3a/fisiologia , Mucinas/metabolismo , Células Th2/fisiologia , Animais , Asma/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Interleucina-13/farmacologia , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Mucina-5ACRESUMO
BACKGROUND: The earliest immune events induced by allergens are poorly understood, yet are likely essential to understanding how allergic inflammation is established. OBJECTIVE: We sought to describe the earliest signaling events activated by allergen and determine their significance to allergic inflammation. METHODS: A fungal-associated allergenic proteinase (FAP) or ovalbumin was administered once intranasally to wild-type mice to determine their ability to induce allergy-associated genes and initiate allergic lung inflammation. Mice deficient in recombinase activating gene 1, C3a, the C3a anaphylatoxin receptor, and MyD88 were challenged similarly to understand the requirement of these molecules and T and B cells for allergic inflammation. Adoptive T-cell transfer experiments were further performed to determine whether signal transducer and activator of transcription 6 (STAT6) was required for cell recruitment and allergic inflammation. RESULTS: FAP, but not ovalbumin, induced eosinophilic airway inflammation and lung IL-4 production in the absence of adaptive immune cells after the transcriptional induction of allergy-specific airway chemokines. Allergen-mediated chemokine secretion and innate allergic lung inflammation occurred in the absence of STAT6, recombinase activating gene 1, C3a, C3a anaphylatoxin receptor, Toll-like receptor 4, and MyD88 but required intact proteinase activity. Furthermore, FAP induced recruitment of T(H)2 cells and eosinophils to lungs independently of STAT6, which was previously thought to be required for T(H)2 cell homing. CONCLUSION: FAP induces allergic lung inflammation through a previously unrecognized innate immune signaling mechanism. CLINICAL IMPLICATIONS: These findings reveal a new paradigm for understanding how allergic inflammation begins and suggest novel possibilities for the prevention and treatment of allergic diseases, such as asthma.
Assuntos
Alérgenos/imunologia , Aspergillus oryzae/enzimologia , Proteínas Fúngicas/imunologia , Hipersensibilidade/complicações , Hipersensibilidade/imunologia , Peptídeo Hidrolases/imunologia , Pneumonia/etiologia , Animais , Quimiocinas/metabolismo , Complemento C3a/metabolismo , Eosinófilos/patologia , Regulação da Expressão Gênica , Hipersensibilidade/genética , Interleucina-4/biossíntese , Pulmão/patologia , Camundongos , Camundongos Endogâmicos , Peptídeo Hidrolases/metabolismo , Pneumonia/patologia , Receptores de Complemento/metabolismo , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Células Th2/patologia , Receptor 4 Toll-Like/metabolismo , Transcrição GênicaRESUMO
RATIONALE: Reports from our laboratory, as well as those from others, have documented the importance of complement activation, the C3a anaphylatoxin, and its receptor, C3aR, in promoting Th2 effector functions in a mouse model of bronchopulmonary allergy. Although deficiency in the fifth complement component (C5) has been linked to enhanced airway hyperresponsiveness in mice, the contribution of C5 to other major biological hallmarks of asthma has not been evaluated. OBJECTIVE: Accordingly, congenic C5-sufficient and C5-deficient mice were subjected to a mouse model of bronchopulmonary allergy to assess the impact of C5 on pulmonary inflammation and Th2 effector functions in experimental asthma. METHODS AND MAIN RESULTS: In contrast to observations reported for C3- and C3aR-deficient animals, C5-deficient mice exhibited significantly increased airway hyperresponsiveness relative to wild-type congenic control mice after antigen challenge. Moreover, challenged C5-deficient mice had a 3.4-fold and 2.7-fold increase in the levels of airway eosinophils and lung interleukin (IL)-4-producing cells, respectively, compared with challenged wild-type mice. Consistent with the numbers of IL-4-producing cells, C5-deficient mice also had increased bronchoalveolar lavage levels of the Th2 cytokines IL-5 and IL-13 and elevated serum levels of total and antigen-specific IgE. CONCLUSIONS: These data indicate that C5 plays an important protective role in allergic lung disease by suppressing inflammatory responses and Th2 effector functions observed in this experimental model. The protection provided by the presence of C5 is likely mediated by C5a, suggesting that C5a may play a significant role in tempering inflammation in Th2-driven diseases such as asthma.
Assuntos
Complemento C5/uso terapêutico , Fatores Imunológicos/uso terapêutico , Hipersensibilidade Respiratória/prevenção & controle , Animais , Biomarcadores/metabolismo , Ativação do Complemento/fisiologia , Complemento C3/deficiência , Complemento C5/deficiência , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Interferon gama/metabolismo , Interleucina-4/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Células Th2/imunologia , Resultado do TratamentoRESUMO
Asthma is a chronic inflammatory disease of the lung resulting in airway obstruction. The airway inflammation of asthma is strongly linked to Th2 lymphocytes and their cytokines, particularly IL-4, IL-5, and IL-13, which regulate airway hyperresponsiveness, eosinophil activation, mucus production, and IgE secretion. Historically, complement was not thought to contribute to the pathogenesis of asthma. However, our previous reports have demonstrated that complement contributes to bronchial hyperreactivity, recruitment of airway eosinophils, IL-4 production, and IgE responses in a mouse model of pulmonary allergy. To define the complement activation fragments that mediate these effects, we assessed the role of the complement anaphylatoxin C3a in a mouse model of pulmonary allergy by challenging C3aR-deficient mice intranasally with a mixed Ag preparation of Aspergillus fumigatus cell culture filtrate and OVA. Analysis by plethysmography after challenge revealed an attenuation in airway hyperresponsiveness in C3aR-deficient mice relative to wild-type mice. C3aR-deficient mice also had an 88% decrease in airway eosinophils and a 59% reduction in lung IL-4-producing cells. Consistent with the reduced numbers of IL-4-producing cells, C3aR-deficient mice had diminished bronchoalveolar lavage levels of the Th2 cytokines, IL-5 and IL-13. C3aR knockout mice also exhibited decreases in IgE titers as well as reduced mucus production. Collectively, these data highlight the importance of complement activation, the C3a anaphylatoxin, and its receptor during Th2 development in this experimental model and implicate these molecules as possible therapeutic targets in diseases such as asthma.
Assuntos
Complemento C3a/metabolismo , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Proteínas de Membrana , Receptores de Complemento/deficiência , Receptores de Complemento/genética , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/imunologia , Células Th2/imunologia , Animais , Antígenos de Fungos/administração & dosagem , Antígenos de Fungos/imunologia , Aspergillus fumigatus/imunologia , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/patologia , Isotipos de Imunoglobulinas/biossíntese , Isotipos de Imunoglobulinas/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Injeções Intraperitoneais , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Interleucina-4/antagonistas & inibidores , Interleucina-4/biossíntese , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucinas/metabolismo , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Receptores de Complemento/fisiologia , Hipersensibilidade Respiratória/metabolismo , Hipersensibilidade Respiratória/patologia , Células Th2/metabolismoRESUMO
Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia, and approximately 80% of patients with cystic fibrosis are infected with this bacterium. To investigate the overall role of complement and the complement activation pathways in the host defense against P. aeruginosa pulmonary infection, we challenged C3-, C4-, and factor B-deficient mice with P. aeruginosa via intranasal inoculation. In these studies, C3(-/-) mice had a higher mortality rate than C3(+/+) mice. Factor B(-/-) mice, but not C4(-/-) mice, infected with P. aeruginosa had a mortality rate similar to that of C3(-/-) mice, indicating that in this model the alternative pathway of complement activation is required for the host defense against Pseudomonas infection. C3(-/-) mice had 6- to 7-fold more bacteria in the lungs and 48-fold more bacteria in the blood than did C3(+/+) mice at 24 h postinfection. In vitro, phagocytic cells from C3(+/+) or C3(-/-) mice exhibited a decreased ability to bind and/or ingest P. aeruginosa in the presence of C3-deficient serum compared to phagocytic cells in the presence of serum with sufficient C3. C3(-/-) mice displayed a significant increase in neutrophils in the lungs and had higher levels of interleukin-1beta (IL-1beta), IL-6, IL-10, KC, and MIP-2 in the lungs at 24 h postinfection than did C3(+/+) mice. Collectively, these results indicate that complement activation by the alternative pathway is critical for the survival of mice infected with P. aeruginosa and that the protection provided by complement is at least in part due to C3-mediated opsonization and phagocytosis of P. aeruginosa.
Assuntos
Complemento C3/metabolismo , Via Alternativa do Complemento , Pneumonia Bacteriana/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa , Animais , Aderência Bacteriana , Atividade Bactericida do Sangue , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocinas/metabolismo , Complemento C3/deficiência , Complemento C3/genética , Complemento C4/deficiência , Complemento C4/genética , Complemento C4/metabolismo , Fator B do Complemento/deficiência , Fator B do Complemento/genética , Fator B do Complemento/metabolismo , Citocinas/metabolismo , Feminino , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Opsonizantes/metabolismo , Fagocitose , Pneumonia Bacteriana/sangue , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/sangue , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/patogenicidadeRESUMO
Mice deficient in complement component C3 (C3(-/-)) and syngeneic C57BL/6 control mice were challenged with Borrelia burgdorferi to determine the role of complement in immune clearance and joint histopathology during experimental Lyme borreliosis. Tibiotarsal joint, ear, and heart tissues were monitored for spirochete numbers at 2, 4, 8, and 12 weeks postinoculation with 10(5) B. burgdorferi B31 clone 5A4 by using quantitative real-time PCR. The spirochete load in joint and ear tissue remained higher in the C3(-/-) mice than in the wild-type counterparts throughout the 12-week study, whereas the numbers in heart tissue of both groups of mice decreased substantially at 8 to 12 weeks postinfection. Histopathology scores for joint tissue were generally higher in the C3(-/-) mice compared to C57BL/6 controls at 2 and 4 weeks postinfection, which may reflect the presence of higher numbers of bacteria in the joints at these early time points. Levels of anti-B. burgdorferi immunoglobulin G tended to be reduced in the C3(-/-) mice compared to control mice. Furthermore, a 5.5-fold-lower number of the complement-sensitive Borrelia garinii was needed to infect C3(-/-) mice compared to C57BL/6 mice, indicating that its sensitivity to complement is one barrier to infection of the mouse model by B. garinii. These results indicate that the complement system may be important in controlling the early dissemination and progression of B. burgdorferi infection.