RESUMO
Background: Sarcoidosis is a chronic inflammatory disease of unknown cause characterized by granuloma formation. Mechanisms for chronic persistence of granulomas are unknown. Matrix Metalloproteinase-12 (MMP12) degrades extracellular matrix elastin and enables infiltration of immune cells responsible for inflammation and granuloma formation. Previous studies report increased MMP12 in sarcoidosis patients and association between MMP12 expression and disease severity. We also observed elevated MMP12 in our multiwall carbon nanotube (MWCNT) murine model of granulomatous inflammation. Here we hypothesized that MMP12 is important to acute and late phases of granuloma pathogenesis. To test this hypothesis, we analyzed granulomatous and inflammatory responses of Mmp12 knock-out (KO) mice at 10 (acute) and 60 days (late) after MWCNT instillation. Methods: C57BL/6 (wildtype) and Mmp12 KO mice underwent oropharyngeal instillation of MWCNT. Lungs were harvested at 3, 10, 20, and 60 days post instillation for evaluation of MMP12 expression and granulomatous changes. Bronchoalveolar lavage (BAL) cells were analyzed 60 days after MWCNT instillation for expression of mediators thought to play a role in sarcoid granulomatosis: peroxisome proliferator-activated receptor-gamma (PPARγ), interferon-gamma (IFN-γ), and CCL2 (MCP-1). Results: Pulmonary granuloma appearance at 10 days after MWCNT instillation showed no differences between wildtype and Mmp12 KO mice. In contrast, by 60 days after MWCNT instillation, Mmp12 KO mice revealed markedly attenuated granuloma formation together with elevated PPARγ and reduced IFNγ expression in BAL cells compared to wildtype. Unexpectedly, Mmp12 KO mice further demonstrated increased alveolar macrophages with increased CCL2 at 60 days. Conclusions: The striking reduction of granuloma formation at day 60 in Mmp12 KO mice suggests that MMP12 is required to maintain chronic granuloma pathophysiology. The increased PPARγ and decreased IFNγ findings suggest that these mediators also may be involved since previous studies have shown that PPARγ suppresses IFNγ and PPARγ deficiency amplifies granuloma formation. Interestingly, a role of MMP12 in granuloma resolution is also suggested by increases in both macrophage influx and CCL2. Overall, our results strongly implicate MMP12 as a key factor in granuloma persistence and as a possible therapeutic target in chronic pulmonary sarcoidosis.
Assuntos
Granuloma/imunologia , Macrófagos Alveolares/imunologia , Metaloproteinase 12 da Matriz/imunologia , Nanotubos de Carbono/efeitos adversos , Sarcoidose Pulmonar/imunologia , Animais , Granuloma/induzido quimicamente , Granuloma/genética , Granuloma/patologia , Macrófagos Alveolares/patologia , Metaloproteinase 12 da Matriz/genética , Camundongos , Camundongos Knockout , Sarcoidose Pulmonar/induzido quimicamente , Sarcoidose Pulmonar/genética , Sarcoidose Pulmonar/patologiaRESUMO
The inflammatory responses in chronic airway diseases leading to emphysema are not fully defined. We hypothesised that lung eosinophilia contributes to airspace enlargement in a mouse model and to emphysema in patients with chronic obstructive pulmonary disease (COPD).A transgenic mouse model of chronic type 2 pulmonary inflammation (I5/hE2) was used to examine eosinophil-dependent mechanisms leading to airspace enlargement. Human sputum samples were collected for translational studies examining eosinophilia and matrix metalloprotease (MMP)-12 levels in patients with chronic airways disease.Airspace enlargement was identified in I5/hE2 mice and was dependent on eosinophils. Examination of I5/hE2 bronchoalveolar lavage identified elevated MMP-12, a mediator of emphysema. We showed, in vitro, that eosinophil-derived interleukin (IL)-13 promoted alveolar macrophage MMP-12 production. Airspace enlargement in I5/hE2 mice was dependent on MMP-12 and eosinophil-derived IL-4/13. Consistent with this, MMP-12 was elevated in patients with sputum eosinophilia and computed tomography evidence of emphysema, and also negatively correlated with forced expiratory volume in 1â s.A mouse model of chronic type 2 pulmonary inflammation exhibited airspace enlargement dependent on MMP-12 and eosinophil-derived IL-4/13. In chronic airways disease patients, lung eosinophilia was associated with elevated MMP-12 levels, which was a predictor of emphysema. These findings suggest an underappreciated mechanism by which eosinophils contribute to the pathologies associated with asthma and COPD.
Assuntos
Eosinófilos/imunologia , Interleucina-13/imunologia , Pneumonia/etiologia , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Enfisema Pulmonar/etiologia , Idoso , Animais , Asma/imunologia , Asma/patologia , Modelos Animais de Doenças , Eosinófilos/patologia , Feminino , Humanos , Interleucina-4/imunologia , Macrófagos Alveolares/patologia , Masculino , Metaloproteinase 12 da Matriz/imunologia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Análise Multivariada , Pneumonia/imunologia , Pneumonia/patologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/patologia , Análise de Regressão , Sistema Respiratório/fisiopatologiaRESUMO
BACKGROUND: Nanomaterials are widely used in various fields. Although the toxicity of carbon nanotubes (CNTs) in pulmonary tissues has been demonstrated, the toxicological effect of CNTs on the immune system in the lung remains unclear. METHODS AND FINDING: In this study, exposure to Taquann-treated multi-walled CNTs (T-CNTs) was performed using aerosols generated in an inhalation chamber. At 12 months after T-CNT exposure, alveolar inflammation with macrophage accumulation and hypertrophy of the alveolar walls were observed. In addition, fibrotic lesions were enhanced by T-CNT exposure. The macrophages in the bronchoalveolar lavage fluid of T-CNT-exposed mice were not largely shifted to any particular population, and were a mixed phenotype with M1 and M2 polarization. Moreover, the alveolar macrophages of T-CNT-exposed mice produced matrix metalloprotinase-12. CONCLUSIONS: These results suggest that T-CNT exposure promoted chronic inflammation and fibrotic lesion formation in profibrotic macrophages for prolonged periods.
Assuntos
Macrófagos Alveolares/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Pneumonia/imunologia , Alvéolos Pulmonares/efeitos dos fármacos , Poluentes Atmosféricos/toxicidade , Poluição do Ar/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Feminino , Fibrose , Humanos , Exposição por Inalação/efeitos adversos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Metaloproteinase 12 da Matriz/imunologia , Metaloproteinase 12 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/induzido quimicamente , Pneumonia/patologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/patologiaRESUMO
Mechanisms driving persistent airway inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. As secretory immunoglobulin A (SIgA) deficiency in small airways has been reported in COPD patients, we hypothesized that immunobarrier dysfunction resulting from reduced SIgA contributes to chronic airway inflammation and disease progression. Here we show that polymeric immunoglobulin receptor-deficient (pIgR(-/-)) mice, which lack SIgA, spontaneously develop COPD-like pathology as they age. Progressive airway wall remodelling and emphysema in pIgR(-/-) mice are associated with an altered lung microbiome, bacterial invasion of the airway epithelium, NF-κB activation, leukocyte infiltration and increased expression of matrix metalloproteinase-12 and neutrophil elastase. Re-derivation of pIgR(-/-) mice in germ-free conditions or treatment with the anti-inflammatory phosphodiesterase-4 inhibitor roflumilast prevents COPD-like lung inflammation and remodelling. These findings show that pIgR/SIgA deficiency in the airways leads to persistent activation of innate immune responses to resident lung microbiota, driving progressive small airway remodelling and emphysema.
Assuntos
Envelhecimento/imunologia , Aminopiridinas/farmacologia , Benzamidas/farmacologia , Microbiota/imunologia , Inibidores da Fosfodiesterase 4/farmacologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Enfisema Pulmonar/imunologia , Receptores de Imunoglobulina Polimérica/deficiência , Envelhecimento/patologia , Remodelação das Vias Aéreas/imunologia , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/imunologia , Ciclopropanos/farmacologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Imunoglobulina A Secretora/genética , Elastase de Leucócito/genética , Elastase de Leucócito/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/microbiologia , Enfisema Pulmonar/tratamento farmacológico , Enfisema Pulmonar/genética , Enfisema Pulmonar/microbiologia , Receptores de Imunoglobulina Polimérica/genética , Receptores de Imunoglobulina Polimérica/imunologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Mucosa Respiratória/patologiaRESUMO
Matrix metalloproteinases (MMPs) are a family of extracellular proteases that play roles in regulating the immune response in inflammatory processes. Previous studies indicated that different MMPs were involved in the host defence and tissue damage in response to different pathogens. However, the contributions of MMPs during Cryptococcus infection have not been addressed clearly. Here, we examined the expression and activity of MMPs during Cryptococcus infection. Among MMP family members, we found significant increases of MMP-3 and MMP-12 mRNA levels and MMP12 zymographic activities in response to C. neoformans but not C. gattii infection. The expression of MMP12 was induced in RAW cells after C. neoformans treatment and in alveolar macrophages purified from C. neoformans-infected mice. Interestingly, administration of MMP inhibitor GM6001 into C. neoformans-infected mice resulted in a significantly increased pulmonary fungal burden with attenuated inflammatory cell infiltration. Corresponding to this finding, the expression of the macrophage- and neutrophil-attracting chemokines CCL2 and CXCL1 was inhibited in the GM6001-treated group and MMP12 levels were found to be correlated strongly with CCL2 mRNA expression. Thus, our data suggest that the induction of MMPs by C. neoformans infection potentiates inflammatory cell infiltration by modulating pulmonary chemokines, thereby promoting effective host immunity to pulmonary Cryptococcus infection.
Assuntos
Quimiocinas/metabolismo , Criptococose/enzimologia , Criptococose/imunologia , Metaloproteinases da Matriz/metabolismo , Pneumonia/imunologia , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Quimiocinas/genética , Quimiocinas/imunologia , Criptococose/microbiologia , Cryptococcus gattii/imunologia , Cryptococcus neoformans/imunologia , Cryptococcus neoformans/patogenicidade , Dipeptídeos/administração & dosagem , Modelos Animais de Doenças , Regulação da Expressão Gênica , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/imunologia , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/imunologia , Inibidores de Metaloproteinases de Matriz/administração & dosagem , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/enzimologia , Pneumonia/microbiologia , Células RAW 264.7RESUMO
Pseudomonas aeruginosa infections persist in patients with cystic fibrosis (CF) and drive lung disease progression. P. aeruginosa potently activates the innate immune system mostly through the recognition of pathogen-associated molecular patterns, such as flagellin. Matrix metalloproteinases 12 and 13 (MMP-12 and MMP-13, respectively) exacerbate chronic lung infection and inflammation by promoting uncontrolled tissue rearrangements and fibrosis, yet the underlying molecular mechanisms by which this occurs remain largely unknown. In this study, we used quantitative bacteriology, histological examination, and proinflammatory cytokine levels to evaluate the effects of MMP-12 and MMP-13 on P. aeruginosa strain K-induced infection and pneumonia in H292 epithelial cells and mice, respectively. Under inflammatory stimulation, mRNA and protein expression levels of proinflammatory mediators were higher in strain K-infected mice and cells than in uninfected counterparts, in which MMP-12 and MMP-13 expression reached levels similar to those observed in epithelial cells. Moreover, we also found that the NF-κB pathway might be involved in the induction of cytokines in response to strain K infection. Taken together, these data suggest that MMP-12 and MMP-13 alter strain K infection in mice and play a role in inflammatory regulation by modulating cytokine levels.
Assuntos
Metaloproteinase 12 da Matriz/imunologia , Metaloproteinase 13 da Matriz/imunologia , Pneumonia/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/patogenicidade , Animais , Linhagem Celular Tumoral , Fibrose Cística/genética , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Citocinas/genética , Citocinas/imunologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 13 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/imunologia , Pneumonia/genética , Pneumonia/microbiologia , Pneumonia/patologia , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/imunologia , Transdução de SinaisRESUMO
Cigarette smoke (CS) is the major cause of chronic obstructive pulmonary disease (COPD). Interleukin-17A (IL-17A) is a pivotal cytokine that regulates lung immunity and inflammation. The aim of the present study was to investigate how IL-17A regulates CS-induced lung inflammation in vivo. IL-17A knockout (KO) mice and neutralization of IL-17A in wild-type (WT) mice reduced macrophage and neutrophil recruitment and chemokine (C-C motif) ligand 2 (CCL2), CCL3 and matrix metalloproteinase (MMP)-12 mRNA expression in response to acute CS exposure. IL-17A expression was increased in non-obese diabetic (NOD) severe combined immunodeficiency SCID) mice with non-functional B- and T-cells over a 4-week CS exposure period, where macrophages accumulated to the same extent as in WT mice. Gene expression analysis by QPCR (quantitative real-time PCR) of isolated immune cell subsets detected increased levels of IL-17A transcript in macrophages, neutrophils and NK/NKT cells in the lungs of CS-exposed mice. In order to further explore the relative contribution of innate immune cellular sources, intracellular IL-17A staining was performed. In the present study, we demonstrate that CS exposure primes natural killer (NK), natural killer T (NKT) and γδ T-cells to produce more IL-17A protein and CS alone increased the frequency of IL17+ γδ T-cells in the lung, whereas IL-17A protein was not detected in macrophages and neutrophils. Our data suggest that activation of innate cellular sources of IL-17A is an essential mediator of macrophage accumulation in CS-exposed lungs. Targeting non-conventional T-cell sources of IL-17A may offer an alternative strategy to reduce pathogenic macrophages in COPD.
Assuntos
Interleucina-17/imunologia , Macrófagos/imunologia , Nicotiana/química , Pneumonia/imunologia , Fumaça , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Quimiocina CCL3/genética , Quimiocina CCL3/imunologia , Quimiocina CCL3/metabolismo , Citometria de Fluxo , Expressão Gênica/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Interleucina-17/genética , Interleucina-17/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/metabolismo , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/imunologia , Metaloproteinase 12 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Infiltração de Neutrófilos/imunologia , Pneumonia/genética , Pneumonia/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Respiratory syncytial virus (RSV) is one of the most frequently observed pathogens during infancy and childhood. However, the corresponding pathogenesis has not been determined to date. We previously demonstrated that IFN-γ plays an important role in RSV pathogenesis, and SARM-TRIF-signaling pathway could regulate the production of IFN-γ. This study is to investigate whether T cells or innate immune cells are the predominant producers of IFN-γ, and further to explore other culprits in addition to IFN-γ in the condition of RSV infection. METHODS: Normal BALB/c mice and nude mice deficient in T cells were infected intranasally with RSV. Leukocytes in bronchoalveolar lavage fluid were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. IFN-γ and MMP-12 were detected by ELISA. MMP408, a selective MMP-12 inhibitor, was given intragastrically. Resveratrol, IFN-γ neutralizing antibody and recombinant murine IFN-γ were administered intraperitoneally. SARM and TRIF protein were semi-quantified by Western blot. siRNA was used to knock-down SARM expression. RESULTS: RSV induced significant airway inflammation and AHR in both mice; IFN-γ was significantly increased in BALB/c mice but not in nude mice. MMP-12 was dramatically increased in both mice but earlier in nude mice. When MMP-12 was inhibited by MMP408, RSV-induced respiratory symptoms were alleviated. SARM was significantly suppressed while TRIF was significantly enhanced in both mice strains. Following resveratrol administration in nude mice, 1) SARM inhibition was prevented, 2) TRIF and MMP-12 were correspondingly down-regulated and 3) airway disorders were subsequently alleviated. Moreover, when SARM was efficiently knocked down using siRNA, TRIF and MMP-12 were markedly enhanced, and the anti-RSV effects of resveratrol were remarkably abrogated. MMP-12 was significantly increased in the IFN-γ neutralizing antibody-treated BALB/c mice but reduced in the recombinant murine IFN-γ-treated nude mice. CONCLUSIONS: MMP-12 can result in at least part of the airway inflammation and AHR independent of IFN-γ. And SARM-TRIF- signaling pathway is involved in regulating the overproduction of MMP-12. To the best of our knowledge, this study is the first that has examined the effects of SARM on MMP-12 and further highlights the potential to target SARM-TRIF-MMP-12 cascades to treat RSV infection.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas do Domínio Armadillo/metabolismo , Hiper-Reatividade Brônquica/enzimologia , Proteínas do Citoesqueleto/metabolismo , Interferon gama/metabolismo , Pulmão/enzimologia , Metaloproteinase 12 da Matriz/metabolismo , Pneumonia/enzimologia , Infecções por Vírus Respiratório Sincicial/enzimologia , Transdução de Sinais , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/imunologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Hiper-Reatividade Brônquica/virologia , Líquido da Lavagem Broncoalveolar/imunologia , Broncoconstrição , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/imunologia , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Imunidade Celular , Interferon gama/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/fisiopatologia , Pulmão/virologia , Metaloproteinase 12 da Matriz/imunologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Pneumonia/genética , Pneumonia/imunologia , Pneumonia/fisiopatologia , Pneumonia/virologia , Interferência de RNA , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/patogenicidade , Linfócitos T/enzimologia , Linfócitos T/imunologia , Linfócitos T/virologia , Fatores de TempoRESUMO
BACKGROUND: Atopic dermatitis (AD) is the most common inflammatory disease. The prevalence of allergic contact dermatitis to allergens (eg, fragrance) is higher in patients with AD, despite a trend toward weaker clinical allergic contact dermatitis reactions. The role of the AD skin phenotype in modulating allergic sensitization to common sensitizers has not been evaluated. OBJECTIVE: We sought to investigate whether patients with AD have altered tissue immune responses on allergen challenge. METHODS: Gene expression and immunohistochemistry studies were performed on biopsy specimens from 10 patients with AD and 14 patients without AD patch tested with common contact allergens (nickel, fragrance, and rubber). RESULTS: Although 1085 differentially expressed genes (DEGs) were commonly modulated in patch-tested skin from patients with AD and patients without AD versus control skin, 1185 DEGs were uniquely altered in skin from patients without AD, and only 246 DEGs were altered in skin from patients with AD. Although many inflammatory products (ie, matrix metalloproteinase 12/matrix metalloproteinase 1/S100A9) were upregulated in both groups, higher-magnitude changes and upregulation of interferon responses were evident only in the non-AD group. Stratification by allergen showed decreased expression of immune, TH1-subset, and TH2-subset genes in nickel-related AD responses, with increased TH17/IL-23 skewing. Rubber/fragrance showed similar trends of lesser magnitude. Negative regulators showed higher expression in patients with AD. CONCLUSIONS: Through contact sensitization, our study offers new insights into AD. Allergic immune reactions were globally attenuated and differentially polarized in patients with AD, with significant decreases in levels of TH1 products, some increases in levels of TH17 products, and inconsistent upregulation in levels of TH2 products. The overall hyporesponsiveness in skin from patients with background AD might be explained by baseline immune abnormalities, such as increased TH2, TH17, and negative regulator levels compared with those seen in non-AD skin.
Assuntos
Alérgenos/imunologia , Citocinas/imunologia , Dermatite Atópica/imunologia , Dermatite de Contato/imunologia , Transcriptoma/imunologia , Adulto , Calgranulina B/genética , Calgranulina B/imunologia , Cosméticos/química , Citocinas/genética , Dermatite Atópica/patologia , Dermatite de Contato/patologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Látex/imunologia , Masculino , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/imunologia , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/imunologia , Pessoa de Meia-Idade , Níquel/imunologia , Testes do Emplastro , Borracha/química , Pele , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologia , Células Th2/imunologia , Células Th2/patologiaRESUMO
Whereas cigarette smoking remains the main risk factor for emphysema, recent studies in ß-epithelial Na(+) channel-transgenic (ßENaC-Tg) mice demonstrated that airway surface dehydration, a key pathophysiological mechanism in cystic fibrosis (CF), caused emphysema in the absence of cigarette smoke exposure. However, the underlying mechanisms remain unknown. The aim of this study was to elucidate mechanisms of emphysema formation triggered by airway surface dehydration. We therefore used expression profiling, genetic and pharmacological inhibition, Foerster resonance energy transfer (FRET)-based activity assays, and genetic association studies to identify and validate emphysema candidate genes in ßENaC-Tg mice and patients with CF. We identified matrix metalloproteinase 12 (Mmp12) as a highly up-regulated gene in lungs from ßENaC-Tg mice, and demonstrate that elevated Mmp12 expression was associated with progressive emphysema formation, which was reduced by genetic deletion and pharmacological inhibition of MMP12 in vivo. By using FRET reporters, we show that MMP12 activity was elevated on the surface of airway macrophages in bronchoalveolar lavage from ßENaC-Tg mice and patients with CF. Furthermore, we demonstrate that a functional polymorphism in MMP12 (rs2276109) was associated with severity of lung disease in CF. Our results suggest that MMP12 released by macrophages activated on dehydrated airway surfaces may play an important role in emphysema formation in the absence of cigarette smoke exposure, and may serve as a therapeutic target in CF and potentially other chronic lung diseases associated with airway mucus dehydration and obstruction.
Assuntos
Obstrução das Vias Respiratórias/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Metaloproteinase 12 da Matriz/imunologia , Muco/imunologia , Enfisema Pulmonar/imunologia , Obstrução das Vias Respiratórias/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Fibrose Cística/genética , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Desidratação/imunologia , Desidratação/metabolismo , Genômica , Macrófagos Alveolares/metabolismo , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/metabolismo , Camundongos Knockout , Muco/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Enfisema Pulmonar/metabolismo , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/imunologia , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais/imunologiaRESUMO
Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. Inflammation and apoptosis have been suggested to be important mechanisms for COPD. Interleukin (IL)-17 superfamily has been associated with chronic inflammation and diminished responses to steroids. It is reasonable to consider that IL-17 may play a role in the pathogenesis of COPD. In this study, we examined IL-17 expression in mice exposed to cigarette smoke (CS) and investigated the contribution of IL-17 to CS-induced inflammation and alveolar cell apoptosis in IL-17(-/-) mice. After exposing wild-type and IL-17(-/-) mice to mainstream CS for 4 wk, IL-17A, but not IL-17F, expression was increased in mice upon CS exposure. Neutrophil infiltration in the lungs of IL-17(-/-) mice was significantly decreased. In IL-17(-/-) mice, there is reduced expression of IL-6, macrophage inflammatory protein-2, and matrix metalloproteinase-12 compared with wild-type mice after CS exposure. The number of apoptotic type II alveolar cells was significantly increased in CS-exposed wild-type mice but not in IL-17(-/-) mice. The effect of IL-17A on type II alveolar cell apoptosis was confirmed in vitro through either addition of IL-17A or transient knockdown of IL-17A by small-interfering RNA transfection in type II alveolar cells. These findings suggest that IL-17A plays an important role in the inflammatory response to CS exposure through increased multiple inflammatory mediators. Moreover, IL-17 may also contribute to type II alveolar cell apoptosis. This study opens a new option in targeting IL-17A to modulate inflammatory response to CS and may be the bases for new therapy for COPD.
Assuntos
Interleucina-17/imunologia , Pneumonia/imunologia , Alvéolos Pulmonares/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Fumar/efeitos adversos , Fumar/imunologia , Animais , Apoptose/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Células Cultivadas , Quimiocina CXCL2/imunologia , Quimiocina CXCL2/metabolismo , Feminino , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Metaloproteinase 12 da Matriz/imunologia , Metaloproteinase 12 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/citologia , Neutrófilos/imunologia , Pneumonia/genética , Pneumonia/patologia , Alvéolos Pulmonares/patologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/citologiaRESUMO
This study investigates the role of proinflammatory monocytes recruited from blood circulation and recovered in bronchoalveolar lavage (BAL) fluid in mediating the lung damage in a model of acute cigarette smoke (CS)-induced lung inflammation in two strains of mice with different susceptibility to develop emphysema (susceptible -C57BL/6J and non susceptible -129S2/SvHsd). Exposure to whole-body CS for 3 consecutive research cigarettes in one single day induced acute inflammation in the lung of mice. Analysis of BAL fluid showed more influx of recently migrated monocytes at 72 h after CS-exposition in susceptible compared to non susceptible mice. It correlated with an increase in MMP-12 and TNF-α protein levels in the lung tissue, and with an increment of NF-κB translocation to the nucleus measured by electrophoretic mobility shift assay in C57BL/6J mice. To determine the functional role of these proinflammatory monocytes in mediating CS-induced airway inflammation, alveolar macrophages and blood monocytes were transiently removed by pretreatment with intratracheal and intravenous liposome-encapsulated CL2MDP, given 2 and 4 days prior to CS exposure and their repopulation was studied. Monocytes/macrophages were maximally depleted 48 h after last liposome application and subsequently recently migrated monocytes reappeared in BAL fluid of susceptible mice at 72 h after CS exposure. Recently migrated monocytes influx to the lung correlated with an increase in the MMP-12 protein level in the lung tissue, indicating that the increase in proinflammatory monocytes is associated with a major tissue damaging. Therefore our data confirm that the recruitment of proinflammatory recently migrated monocytes from the blood are responsible for the increase in MMP-12 and has an important role in the pathogenesis of lung disease induced by acute lung inflammation. These results could contribute to understanding the different susceptibility to CS of these strains of mice.
Assuntos
Movimento Celular/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Nicotiana/efeitos adversos , Enfisema Pulmonar/patologia , Fumaça/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Movimento Celular/imunologia , Ácido Clodrônico/administração & dosagem , Expressão Gênica , Lipossomos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Masculino , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/imunologia , Monócitos/patologia , NF-kappa B/genética , NF-kappa B/imunologia , Pneumonia/etiologia , Pneumonia/imunologia , Pneumonia/patologia , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/imunologia , Especificidade da Espécie , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Although macrophages are widely recognized to have a profibrotic role in inflammation, we have used a highly tractable CCl(4)-induced model of reversible hepatic fibrosis to identify and characterize the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11B(hi) F4/80(int) Ly-6C(lo) macrophage subset was most abundant in livers during maximal fibrosis resolution and represented the principle matrix metalloproteinase (MMP) -expressing subset. Depletion of this population in CD11B promoter-diphtheria toxin receptor (CD11B-DTR) transgenic mice caused a failure of scar remodeling. Adoptive transfer and in situ labeling experiments showed that these restorative macrophages derive from recruited Ly-6C(hi) monocytes, a common origin with profibrotic Ly-6C(hi) macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6C(lo) subset, compared with Ly-6C(hi) macrophages, showed a phenotype outside the M1/M2 classification, with increased expression of MMPs, growth factors, and phagocytosis-related genes, including Mmp9, Mmp12, insulin-like growth factor 1 (Igf1), and Glycoprotein (transmembrane) nmb (Gpnmb). Confocal microscopy confirmed the postphagocytic nature of restorative macrophages. Furthermore, the restorative macrophage phenotype was recapitulated in vitro by the phagocytosis of cellular debris with associated activation of the ERK signaling cascade. Critically, induced phagocytic behavior in vivo, through administration of liposomes, increased restorative macrophage number and accelerated fibrosis resolution, offering a therapeutic strategy to this orphan pathological process.
Assuntos
Antígenos Ly/imunologia , Intoxicação por Tetracloreto de Carbono/imunologia , Regulação da Expressão Gênica/imunologia , Cirrose Hepática/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Animais , Antígenos Ly/genética , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Tetracloreto de Carbono/toxicidade , Intoxicação por Tetracloreto de Carbono/genética , Intoxicação por Tetracloreto de Carbono/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/imunologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/patologia , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/imunologia , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Transgênicos , Monócitos/patologiaRESUMO
BACKGROUND: Matrix metalloproteinase (MMP)-12 has been implicated in the pathogenesis of both chronic obstructive pulmonary disease (COPD) and asthma. The influence of disease severity on sputum MMP-12 concentrations and activity is not known. OBJECTIVES: We sought to examine the relationship between disease severity assessed by means of lung function and computed tomography (CT) and induced sputum MMP-12 concentrations and activity in patients with asthma and COPD. METHODS: In 208 subjects (109 asthmatic patients, smokers and never smokers, mild, moderate, and severe; 53 patients with COPD, smokers and exsmokers, mild, moderate, and severe; and 46 healthy control subjects, smokers and never smokers), we measured induced sputum MMP-12 concentrations (ELISA) and enzyme activity (fluorescence resonance energy transfer), sputum cell MMP12 mRNA expression (quantitative PCR [qPCR]), diffusing capacity for carbon monoxide (Dlco), and CT assessment of emphysema (percentage of low-attenuation areas at less -950 Hounsfield units). RESULTS: Sputum MMP-12 concentrations are greater in patients with COPD and smokers with asthma than in healthy nonsmokers (P = .003 and P = .035, respectively) but similar to those seen in healthy smokers. In patients with COPD, disease severity, when measured by means of CT-assessed emphysema, but not by means of spirometry or Dlco values, is directly associated with sputum MMP-12 concentrations and activity. In the asthma groups there is no significant association between disease severity and sputum MMP-12 concentrations or activity. CONCLUSIONS: Sputum MMP-12 concentrations and activity in patients with COPD are directly associated with the extent of emphysema measured by means of CT. This finding supports a role for MMP-12 in the pathogenesis of COPD and might suggest that blocking MMP-12 activity in patients with COPD could prevent the further development of emphysema.
Assuntos
Asma/imunologia , Asma/fisiopatologia , Metaloproteinase 12 da Matriz/metabolismo , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Escarro/enzimologia , Adulto , Idoso , Asma/complicações , Asma/diagnóstico , Estudos Transversais , Progressão da Doença , Enfisema/diagnóstico , Enfisema/enzimologia , Feminino , Transferência Ressonante de Energia de Fluorescência , Seguimentos , Humanos , Masculino , Metaloproteinase 12 da Matriz/genética , Metaloproteinase 12 da Matriz/imunologia , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Testes de Função Respiratória , Índice de Gravidade de Doença , Tomografia Computadorizada por Raios XRESUMO
Matrix metalloproteinase 12 (MMP12) is a macrophage-secreting proteinase. To fully understand the function of MMP12 in myeloid lineage cells, a myeloid-specific c-fms-rtTA/(TetO)7-CMV-MMP12 bitransgenic mouse model was created. In this bitransgenic system, induction of MMP12 abnormally elevated frequencies and numbers of common myeloid progenitor (CMP) and granulocyte/macrophage progenitor (GMP) populations, and decreased the frequency and number of the megakaryocyte/erythrocyte progenitor (MEP) population in the bone marrow (BM). The CD11b(+)/Gr-1(+) immature cell population was systemically increased in multiple organs. Both in vitro and in vivo studies showed an immunosuppressive function on T-cell proliferation and function by CD11b(+)/Gr-1(+) immature cells from MMP12-overexpressing bitransgenic mice. MMP12 directly stimulated lineage-negative (Linâ») progenitor cells to differentiate into CD11b(+)/Gr-1(+) immature cells that showed immunosuppression on T-cell proliferation and function in vitro. Regulatory T cells (Tregs) were increased. In the lung, the concentration of IL-6 was increased, which aberrantly activated oncogenic Stat3 and increased expression of Stat3 downstream genes in epithelial tumor progenitor cells. Spontaneous emphysema and lung adenocarcinoma were sequentially developed after MMP12 overexpression. BM chimeras confirmed that the MMP12-induced myeloid cell autonomous defect led to abnormal myelopoiesis, immune suppression, and lung adenocarcinoma.
Assuntos
Adenocarcinoma , Células-Tronco Hematopoéticas , Tolerância Imunológica/fisiologia , Neoplasias Pulmonares , Metaloproteinase 12 da Matriz/genética , Células Mieloides , Adenocarcinoma/imunologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Antígeno CD11b/metabolismo , Diferenciação Celular/imunologia , Linhagem Celular , Regulação Enzimológica da Expressão Gênica/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metaloproteinase 12 da Matriz/imunologia , Metaloproteinase 12 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Células Mieloides/enzimologia , Células Mieloides/imunologia , Células Mieloides/patologia , Alvéolos Pulmonares/citologia , Receptores de Superfície Celular/metabolismo , Mucosa Respiratória/enzimologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Transdução de Sinais/imunologia , Linfócitos T/citologia , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Proteolysis of matrix components, in particular elastin, is a major contributing factor to the development of lung diseases such as emphysema and chronic obstructive pulmonary disease (COPD). MMP-12 (macrophage elastase) is a protease known to be involved in the progression of lung disease. The relatively low abundance of MMP-12 has precluded the development of quantitative assays that can accurately measure MMP-12 protein levels and activity across cohorts of healthy and diseased individuals. METHODS: Commercial antibodies were screened for performance in sandwich ELISA and capture FRET activity assay formats. Precision, accuracy, sensitivity, dilution linearity, and spike recovery were evaluated using sputum samples. RESULTS: Total protein and capture FRET activity assays were developed that were sensitive enough to detect MMP-12 in 37 of 38 donor sputum samples. A comparison of results between the two assays shows that a majority of sputum MMP-12 is in the active form. No differences were seen between normal, asthmatic, and COPD donors. CONCLUSION: Sensitive and quantitative assays for both MMP-12 activity and total protein in human induced sputum have been developed. These assays can be used to evaluate MMP-12 as a biomarker for lung disease, and to monitor efficacy of potential therapeutic compounds.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Metaloproteinase 12 da Matriz/metabolismo , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Escarro/enzimologia , Especificidade de Anticorpos , Calibragem , Elastina/metabolismo , Ensaio de Imunoadsorção Enzimática/normas , Transferência Ressonante de Energia de Fluorescência/normas , Humanos , Técnicas de Diluição do Indicador , Metaloproteinase 12 da Matriz/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Formation of osteoclasts and consequent joint destruction are hallmarks of rheumatoid arthritis (RA). Here we show that LIGHT, a member of the tumour necrosis factor (TNF) superfamily, induced the differentiation into tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) of CD14(+) monocytes cocultured with nurse-like cells isolated from RA synovium, but not of freshly isolated CD14(+) monocytes. Receptor activator of nuclear factor-kappaB ligand (RANKL) enhanced this LIGHT-induced generation of TRAP-positive MNCs. The MNCs showed the phenotypical and functional characteristics of osteoclasts; they showed the expression of osteoclast markers such as cathepsin K, actin-ring formation, and the ability to resorb bone. Moreover, the MNCs expressed both matrix metalloproteinase 9 (MMP-9) and MMP-12, but the latter was not expressed in osteoclasts induced from CD14(+) monocytes by RANKL. Immunohistochemical analysis showed that the MMP-12-producing MNCs were present in the erosive areas of joints in RA, but not in the affected joints of osteoarthritic patients. These findings suggested that LIGHT might be involved in the progression of inflammatory bone destruction in RA, and that osteoclast progenitors might become competent for LIGHT-mediated osteoclastogenesis via interactions with synoviocyte-like nurse-like cells.