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1.
Cell ; 180(3): 490-501.e16, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31955848

RESUMO

Integrin αvß8 binds with exquisite specificity to latent transforming growth factor-ß (L-TGF-ß). This binding is essential for activating L-TGF-ß presented by a variety of cell types. Inhibiting αvß8-mediated TGF-ß activation blocks immunosuppressive regulatory T cell differentiation, which is a potential therapeutic strategy in cancer. Using cryo-electron microscopy, structure-guided mutagenesis, and cell-based assays, we reveal the binding interactions between the entire αvß8 ectodomain and its intact natural ligand, L-TGF-ß, as well as two different inhibitory antibody fragments to understand the structural underpinnings of αvß8 binding specificity and TGF-ß activation. Our studies reveal a mechanism of TGF-ß activation where mature TGF-ß signals within the confines of L-TGF-ß and the release and diffusion of TGF-ß are not required. The structural details of this mechanism provide a rational basis for therapeutic strategies to inhibit αvß8-mediated L-TGF-ß activation.


Assuntos
Microscopia Crioeletrônica/métodos , Integrinas/química , Integrinas/metabolismo , Proteínas de Ligação a TGF-beta Latente/química , Proteínas de Ligação a TGF-beta Latente/metabolismo , Fator de Crescimento Transformador beta1/química , Fator de Crescimento Transformador beta1/metabolismo , Animais , Anticorpos/imunologia , Sítios de Ligação , Brônquios/citologia , Células CHO , Cricetulus , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Integrinas/imunologia , Ativação Linfocitária , Masculino , Vison , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Linfócitos T Reguladores/imunologia
2.
Circulation ; 143(9): 935-948, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33307764

RESUMO

BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.


Assuntos
Cadeias beta de Integrinas/química , Compostos de Sulfidrila/química , Animais , Cromatografia Líquida de Alta Pressão , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cisteína/química , Dissulfetos/análise , Dissulfetos/química , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Sulfeto de Hidrogênio/farmacologia , Cadeias beta de Integrinas/metabolismo , Mecanotransdução Celular , Camundongos , Resistência ao Cisalhamento , Espectrometria de Massas em Tandem , Vasodilatação/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo
3.
Emerg Radiol ; 29(3): 611-614, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35128620

RESUMO

In this case report, dual-energy CT was critical in the diagnosis of acute mesenteric ischemia by differentiating normal contrast-enhanced bowel and hemorrhagic necrosis. Iodine map showed a segment of small bowel with minimal contrast enhancement, and virtual non-contrast imaging revealed hyperattenuating bowel. This finding changed management for the patient and prevented complications from impending bowel perforation. Histopathological analysis confirmed hemorrhagic necrosis of the bowel segment. In cases of suspected bowel ischemia, dual-energy CT can distinguish bowel wall hemorrhage from contrast enhancement and allow for accurate diagnosis.


Assuntos
Iodo , Isquemia Mesentérica , Meios de Contraste , Hemorragia Gastrointestinal , Humanos , Intestino Delgado , Isquemia , Isquemia Mesentérica/diagnóstico por imagem , Necrose/complicações , Necrose/patologia , Tomografia Computadorizada por Raios X/métodos
4.
Proc Natl Acad Sci U S A ; 114(47): E10161-E10168, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29109269

RESUMO

Human regulatory T cells (Tregs) suppress other T cells by converting the latent, inactive form of TGF-ß1 into active TGF-ß1. In Tregs, TGF-ß1 activation requires GARP, a transmembrane protein that binds and presents latent TGF-ß1 on the surface of Tregs stimulated through their T cell receptor. However, GARP is not sufficient because transduction of GARP in non-Treg T cells does not induce active TGF-ß1 production. RGD-binding integrins were shown to activate TGF-ß1 in several non-T cell types. Here we show that αVß8 dimers are present on stimulated human Tregs but not in other T cells, and that antibodies against αV or ß8 subunits block TGF-ß1 activation in vitro. We also show that αV and ß8 interact with GARP/latent TGF-ß1 complexes in human Tregs. Finally, a blocking antibody against ß8 inhibited immunosuppression by human Tregs in a model of xenogeneic graft-vs.-host disease induced by the transfer of human T cells in immunodeficient mice. These results show that TGF-ß1 activation on the surface of human Tregs implies an interaction between the integrin αVß8 and GARP/latent TGF-ß1 complexes. Immunosuppression by human Tregs can be inhibited by antibodies against GARP or against the integrin ß8 subunit. Such antibodies may prove beneficial against cancer or chronic infections.


Assuntos
Doença Enxerto-Hospedeiro/imunologia , Tolerância Imunológica/efeitos dos fármacos , Integrinas/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta1/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Células Cultivadas , Modelos Animais de Doenças , Humanos , Integrinas/antagonistas & inibidores , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T Reguladores/transplante , Fator de Crescimento Transformador beta1/metabolismo , Transplante Heterólogo
5.
Am J Physiol Lung Cell Mol Physiol ; 315(1): L25-L40, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29543040

RESUMO

Evidence is accumulating that exposure to cigarette smoke (CS) increases the risk of developing acute respiratory distress syndrome (ARDS). Streptococcus pneumoniae is the most common cause of bacterial pneumonia, which in turn is the leading cause of ARDS. Chronic smokers have increased rates of pneumococcal colonization and develop more severe pneumococcal pneumonia than nonsmokers; yet mechanistic connections between CS exposure, bacterial pneumonia, and ARDS pathogenesis remain relatively unexplored. We exposed mice to 3 wk of moderate whole body CS or air, followed by intranasal inoculation with an invasive serotype of S. pneumoniae. CS exposure alone caused no detectable lung injury or bronchoalveolar lavage (BAL) inflammation. During pneumococcal infection, CS-exposed mice had greater survival than air-exposed mice, in association with reduced systemic spread of bacteria from the lungs. However, when mice were treated with antibiotics after infection to improve clinical relevance, the survival benefit was lost, and CS-exposed mice had more pulmonary edema, increased numbers of BAL monocytes, and elevated monocyte and lymphocyte chemokines. CS-exposed antibiotic-treated mice also had higher serum surfactant protein D and angiopoietin-2, consistent with more severe lung epithelial and endothelial injury. The results indicate that acute CS exposure enhances the recruitment of immune cells to the lung during bacterial pneumonia, an effect that may provide microbiological benefit but simultaneously exposes the mice to more severe inflammatory lung injury. The inclusion of antibiotic treatment in preclinical studies of acute lung injury in bacterial pneumonia may enhance clinical relevance, particularly for future studies of current or emerging tobacco products.


Assuntos
Lesão Pulmonar Aguda , Antibacterianos/farmacologia , Pneumonia Bacteriana , Pneumonia Pneumocócica , Streptococcus pneumoniae/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Feminino , Camundongos , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/metabolismo , Pneumonia Bacteriana/patologia , Pneumonia Pneumocócica/tratamento farmacológico , Pneumonia Pneumocócica/metabolismo , Pneumonia Pneumocócica/patologia , Edema Pulmonar/tratamento farmacológico , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia
6.
Am J Physiol Lung Cell Mol Physiol ; 312(1): L122-L130, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27913421

RESUMO

Small airway fibrosis is a major pathological feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Chronic inflammatory cells accumulate around small airways in COPD and are thought to play a major role in small airway fibrosis. Mice deficient in α/ß T cells have recently been shown to be protected from both experimental airway inflammation and fibrosis. In these models, CD4+Th17 cells and secretion of IL-17A are increased. However, a pathogenic role for IL-17 in specifically mediating fibrosis around airways has not been demonstrated. Here a role for IL-17A in airway fibrosis was demonstrated using mice deficient in the IL-17 receptor A (il17ra) Il17ra-deficient mice were protected from both airway inflammation and fibrosis in two different models of airway fibrosis that employ COPD-relevant stimuli. In these models, CD4+ Th17 are a major source of IL-17A with other expressing cell types including γδ T cells, type 3 innate lymphoid cells, polymorphonuclear cells, and CD8+ T cells. Antibody neutralization of IL-17RA or IL-17A confirmed that IL-17A was the relevant pathogenic IL-17 isoform and IL-17RA was the relevant receptor in airway inflammation and fibrosis. These results demonstrate that the IL-17A/IL-17 RA axis is crucial to murine airway fibrosis. These findings suggest that IL-17 might be targeted to prevent the progression of airway fibrosis in COPD.


Assuntos
Interleucina-17/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Adenoviridae/metabolismo , Animais , Modelos Animais de Doenças , Interleucina-1beta/farmacologia , Camundongos Endogâmicos C57BL , Testes de Neutralização , Pneumonia/complicações , Pneumonia/metabolismo , Pneumonia/patologia , Poli I-C/farmacologia , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/patologia , Fibrose Pulmonar/complicações , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Receptores de Interleucina-17/metabolismo , Fumar/efeitos adversos
7.
J Immunol ; 194(8): 3962-9, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25786688

RESUMO

Chronic airway inflammation and fibrosis, known as airway remodeling, are defining features of chronic obstructive pulmonary disease and are refractory to current treatments. How and whether chronic inflammation contributes to airway fibrosis remain controversial. In this study, we use a model of chronic obstructive pulmonary disease airway disease utilizing adenoviral delivery of IL-1ß to determine that adaptive T cell immunity is required for airway remodeling because mice deficient in α/ß T cells (tcra(-/-)) are protected. Dendritic cells (DCs) accumulate around chronic obstructive pulmonary disease airways and are critical to prime adaptive immunity, but they have not been shown to directly influence airway remodeling. We show that DC depletion or deficiency in the crucial DC chemokine receptor ccr6 both protect from adenoviral IL-1ß-induced airway adaptive T cell immune responses and fibrosis in mice. These results provide evidence that chronic airway inflammation, mediated by accumulation of α/ß T cells and driven by DCs, is critical to airway fibrosis.


Assuntos
Imunidade Adaptativa , Células Dendríticas/imunologia , Interleucina-1beta/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Fibrose Pulmonar/imunologia , Animais , Células Dendríticas/patologia , Interleucina-1beta/genética , Camundongos , Camundongos Knockout , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Fibrose Pulmonar/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia
8.
J Immunol ; 195(3): 1182-90, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26109638

RESUMO

Small airway chronic inflammation is a major pathologic feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Dendritic cells (DCs) accumulate around small airways in COPD. DCs are critical mediators of Ag surveillance and Ag presentation and amplify adaptive immune responses. How DCs accumulate around airways remains largely unknown. We use 2-photon DC imaging of living murine lung sections to directly visualize the dynamic movement of living DCs around airways in response to either soluble mediators (IL-1ß) or environmental stimuli (cigarette smoke or TLR3 ligands) implicated in COPD pathogenesis. We find that DCs accumulate around murine airways primarily by increasing velocity (chemokinesis) rather than directional migration (chemotaxis) in response to all three stimuli. DC accumulation maximally occurs in a specific zone located 26-50 µm from small airways, which overlaps with zones of maximal DC velocity. Our data suggest that increased accumulation of DCs around airways results from increased numbers of highly chemokinetic DCs entering the lung from the circulation with balanced rates of immigration and emigration. Increases in DC accumulation and chemokinesis are partially dependent on ccr6, a crucial DC chemokine receptor, and fibroblast expression of the integrin αvß8, a critical activator of TGF-ß. αvß8-Mediated TGF-ß activation is known to enhance IL-1ß-dependent fibroblast expression of the only known endogenous ccr6 chemokine ligand, ccl20. Taken together, these data suggest a mechanism by which αvß8, ccl20, and ccr6 interact to lead to DC accumulation around airways in response to COPD-relevant stimuli.


Assuntos
Células Dendríticas/imunologia , Integrinas/imunologia , Interleucina-1beta/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Fator de Crescimento Transformador beta/imunologia , Imunidade Adaptativa/imunologia , Animais , Movimento Celular/imunologia , Quimiocina CCL20/biossíntese , Quimiocina CCL20/imunologia , Modelos Animais de Doenças , Ativação Enzimática/imunologia , Fibroblastos/imunologia , Integrinas/biossíntese , Interleucina-1beta/biossíntese , Pulmão/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli I-C/farmacologia , Doença Pulmonar Obstrutiva Crônica/patologia , Radiografia , Receptores CCR6/genética , Receptores CCR6/imunologia , Fumaça/efeitos adversos , Receptor 3 Toll-Like , Fator de Crescimento Transformador beta/metabolismo
9.
Nicotine Tob Res ; 19(9): 1033-1039, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28340238

RESUMO

INTRODUCTION: Cigarette smoking (CS) remains a major public health concern and has recently been associated with an increased risk of developing acute respiratory distress syndrome (ARDS). Bronchoalveolar lavage (BAL) experiments in human volunteers have demonstrated that active smokers develop increased alveolar-epithelial barrier permeability to protein after inhaling lipopolysaccharide (LPS). Here we tested the hypothesis that short-term whole-body CS exposure would increase LPS-induced lung edema in mice. METHODS: Adult mice were exposed in a Teague TE-10 machine to CS from 3R4F cigarettes at 100 mg/m3 total suspended particulates for 12 days, then given LPS or saline intratracheally. Control mice were housed in the same room without CS exposure. Post-mortem measurements included gravimetric lung water and BAL protein, cell counts, and lung histology. Cytokines were measured in lung homogenate by ELISA and in plasma by Luminex and ELISA. RESULTS: In CS-exposed mice, intratracheal LPS caused greater increases in pulmonary edema by gravimetric measurement and histologic scoring. CS-exposed mice also had an increase in BAL neutrophilia, lung IL-6, and plasma CXCL9, a T-cell chemoattractant. Intratracheal LPS concentrated blood hemoglobin to a greater degree in CS-exposed mice, consistent with an increase in systemic vascular permeability. CONCLUSIONS: These results demonstrate that CS exposure in endotoxin injured mice increases the severity of acute lung injury. The increased lung IL-6 in CS-exposed LPS-injured mice indicates that this potent cytokine, previously shown to predict mortality in patients with ARDS, may play a role in exacerbating lung injury in smokers and may have utility as a biomarker of tobacco-related lung injury. IMPLICATIONS: Our results suggest that short-term CS exposure at levels that cause no overt lung injury may still prime the lung for acute inflammatory damage from a "second hit", a finding that mirrors the increased risk of developing ARDS in patients who smoke. This model may be useful for evaluating the acute pulmonary toxicity of existing and/or novel tobacco products and identifying biomarkers of tobacco-related lung injury.


Assuntos
Lipopolissacarídeos/efeitos adversos , Lesão Pulmonar , Pulmão , Edema Pulmonar , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/fisiopatologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/imunologia , Lesão Pulmonar/fisiopatologia , Camundongos , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/imunologia , Edema Pulmonar/fisiopatologia , Síndrome do Desconforto Respiratório
10.
J Biol Chem ; 290(23): 14717-28, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25918170

RESUMO

CCL20 is the only chemokine ligand for the chemokine receptor CCR6, which is expressed by the critical antigen presenting cells, dendritic cells. Increased expression of CCL20 is likely involved in the increased recruitment of dendritic cells observed in fibroinflammatory diseases such as chronic obstructive pulmonary disease (COPD). CCL20 expression is increased by the proinflammatory cytokine IL-1ß. We have determined that IL-1ß-dependent CCL20 expression is also dependent on the multifunctional cytokine TGF-ß. TGF-ß is expressed in a latent form that must be activated to function, and activation is achieved through binding to the integrin αvß8 (itgb8). Here we confirm correlative increases in αvß8 and IL-1ß with CCL20 protein in lung parenchymal lysates of a large cohort of COPD patients. How IL-1ß- and αvß8-mediated TGF-ß activation conspire to increase fibroblast CCL20 expression remains unknown, because these pathways have not been shown to directly interact. We evaluate the 5'-flanking region of CCL20 to determine that IL-1ß-driven CCL20 expression is dependent on αvß8-mediated activation of TGF-ß. We identify a TGF-ß-responsive element (i.e. SMAD) located on an upstream enhancer of the human CCL20 promoter required for efficient IL-1ß-dependent CCL20 expression. By chromatin immunoprecipitation, this upstream enhancer complexes with the p50 subunit of NF-κB on a NF-κB-binding element close to the transcriptional start site of CCL20. These interactions are confirmed by electromobility shift assays in nuclear extracts from human lung fibroblasts. These data define a mechanism by which αvß8-dependent activation of TGF-ß regulates IL-1ß-dependent CCL20 expression in COPD.


Assuntos
Quimiocina CCL20/genética , Interleucina-1beta/imunologia , Elementos de Resposta , Transdução de Sinais , Fator de Crescimento Transformador beta/imunologia , Animais , Sequência de Bases , Células Cultivadas , Fibroblastos/imunologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia
11.
J Immunol ; 192(3): 958-68, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24367027

RESUMO

Cigarette smoke (CS)-induced cellular senescence has been implicated in the pathogenesis of chronic obstructive pulmonary disease, and SIRT6, a histone deacetylase, antagonizes this senescence, presumably through the attenuation of insulin-like growth factor (IGF)-Akt signaling. Autophagy controls cellular senescence by eliminating damaged cellular components and is negatively regulated by IGF-Akt signaling through the mammalian target of rapamycin (mTOR). SIRT1, a representative sirtuin family, has been demonstrated to activate autophagy, but a role for SIRT6 in autophagy activation has not been shown. Therefore, we sought to investigate the regulatory role for SIRT6 in autophagy activation during CS-induced cellular senescence. SIRT6 expression levels were modulated by cDNA and small interfering RNA transfection in human bronchial epithelial cells (HBECs). Senescence-associated ß-galactosidase staining and Western blotting of p21 were performed to evaluate senescence. We demonstrated that SIRT6 expression levels were decreased in lung homogenates from chronic obstructive pulmonary disease patients, and SIRT6 expression levels correlated significantly with the percentage of forced expiratory volume in 1 s/forced vital capacity. CS extract (CSE) suppressed SIRT6 expression in HBECs. CSE-induced HBEC senescence was inhibited by SIRT6 overexpression, whereas SIRT6 knockdown and mutant SIRT6 (H133Y) without histone deacetylase activity enhanced HBEC senescence. SIRT6 overexpression induced autophagy via attenuation of IGF-Akt-mTOR signaling. Conversely, SIRT6 knockdown and overexpression of a mutant SIRT6 (H133Y) inhibited autophagy. Autophagy inhibition by knockdown of ATG5 and LC3B attenuated the antisenescent effect of SIRT6 overexpression. These results suggest that SIRT6 is involved in CSE-induced HBEC senescence via autophagy regulation, which can be attributed to attenuation of IGF-Akt-mTOR signaling.


Assuntos
Autofagia/fisiologia , Brônquios/patologia , Senescência Celular/fisiologia , Células Epiteliais/patologia , Fator de Crescimento Insulin-Like I/fisiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Sirtuínas/fisiologia , Fumaça/efeitos adversos , Acetilação , Proteína 5 Relacionada à Autofagia , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Volume Expiratório Forçado , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/fisiologia , Mutação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/fisiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/fisiologia , Sirtuínas/antagonistas & inibidores , Sirtuínas/genética , Serina-Treonina Quinases TOR/fisiologia , Nicotiana , Capacidade Vital
12.
Am J Physiol Lung Cell Mol Physiol ; 304(1): L56-69, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23087019

RESUMO

Autophagy, a process that helps maintain homeostatic balance between the synthesis, degradation, and recycling of organelles and proteins to meet metabolic demands, plays an important regulatory role in cellular senescence and differentiation. Here we examine the regulatory role of autophagy in idiopathic pulmonary fibrosis (IPF) pathogenesis. We test the hypothesis that epithelial cell senescence and myofibroblast differentiation are consequences of insufficient autophagy. Using biochemical evaluation of in vitro models, we find that autophagy inhibition is sufficient to induce acceleration of epithelial cell senescence and myofibroblast differentiation in lung fibroblasts. Immunohistochemical evaluation of human IPF biospecimens reveals that epithelial cells show increased cellular senescence, and both overlaying epithelial cells and fibroblasts in fibroblastic foci (FF) express both ubiquitinated proteins and p62. These findings suggest that insufficient autophagy is an underlying mechanism of both accelerated cellular senescence and myofibroblast differentiation in a cell-type-specific manner and is a promising clue for understanding the pathogenesis of IPF.


Assuntos
Autofagia , Fibrose Pulmonar Idiopática/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Diferenciação Celular/fisiologia , Senescência Celular/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Humanos , Miofibroblastos/citologia , Proteína Sequestossoma-1 , Tunicamicina/farmacologia , Ubiquitina/biossíntese
13.
J Immunol ; 187(1): 510-9, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21646299

RESUMO

TLR3, one of the TLRs involved in the recognition of infectious pathogens for innate and adaptive immunity, primarily recognizes viral-associated dsRNA. Recognition of dsRNA byproducts released from apoptotic and necrotic cells is a recently proposed mechanism for the amplification of toxicity, suggesting a pivotal participation of TLR3 in viral infection, as well as in lung diseases where apoptosis plays a critical role, such as asthma and chronic obstructive pulmonary disease. In addition to metabolic control, insulin signaling was postulated to be protective by inhibiting apoptosis. Therefore, we explored the role of insulin signaling in protecting against TLR3-mediated apoptosis of human bronchial epithelial cells. Significant TLR3-mediated apoptosis was induced by polyinosinic-polycytidylic acid, a dsRNA analog, via caspase-8-dependent mechanisms. However, insulin efficiently inhibited TLR3/polyinosinic-polycytidylic acid-induced human bronchial epithelial cell apoptosis via PI3K/Akt and ERK pathways, at least in part, via upregulation of cellular FLIPs and through protein synthesis-independent mechanisms. These results indicate the significance of TLR3-mediated dsRNA-induced apoptosis in the pathogenesis of apoptosis-driven lung disease and provide evidence for a novel protective role of insulin.


Assuntos
Apoptose/imunologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Insulina/fisiologia , Sistema de Sinalização das MAP Quinases/imunologia , Fosfatidilinositol 3-Quinase/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Mucosa Respiratória/imunologia , Receptor 3 Toll-Like/antagonistas & inibidores , Brônquios/enzimologia , Brônquios/imunologia , Brônquios/patologia , Células Cultivadas , Humanos , Mucosa Respiratória/enzimologia , Mucosa Respiratória/patologia , Receptor 3 Toll-Like/fisiologia
14.
J Biol Chem ; 286(42): 36864-74, 2011 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-21878622

RESUMO

The integrin αvß8 is a cell surface receptor for the latent domain (LAP) of the multifunctional cytokine TGF-ß. Through its association with LAP, TGF-ß is maintained in a latent form that must be activated to function. Binding to the integrin αvß8 with subsequent metalloproteolytic cleavage of LAP represents a major mechanism of TGF-ß activation in vivo. Altered expression of the integrin ß8 subunit (ITGB8) is found in human chronic obstructive pulmonary disease, cancers, and brain vascular malformations. We have previously shown that the proinflammatory cytokine interleukin-1ß (IL-1ß) increases ITGB8 expression on lung fibroblasts, which increases αvß8-mediated TGF-ß activation in fibrosis and pathologic inflammation. Here we report the mechanism of increased ITGB8 expression by IL-1ß. Our data support a model where the chromatin architecture of the ITGB8 core promoter is altered by nucleosomal repositioning that enhances the interaction of an AP1 complex (containing c-Jun and ATF2). This repositioning is caused by the dissociation of HDAC2 with the ITGB8 core promoter, leading to increased histone H4 acetylation and a loosening of nucleosomal-DNA interactions allowing "opening" of the chromatin structure and increased association of c-Jun and ATF-2. These changes are mediated through NFκB- and p38-dependent pathways. Ultimately, these events culminate in increasing ITGB8 transcription, αvß8 surface expression, and αvß8-mediated TGFß activation.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Cadeias beta de Integrinas/biossíntese , Interleucina-1beta/biossíntese , Regiões Promotoras Genéticas , Fator de Crescimento Transformador beta/metabolismo , Acetilação , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , DNA/genética , DNA/metabolismo , Células HeLa , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Integrina alfa5/biossíntese , Integrina alfa5/genética , Cadeias beta de Integrinas/genética , Interleucina-1beta/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Crescimento Transformador beta/genética
16.
Proc Natl Acad Sci U S A ; 106(48): 20464-9, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19920174

RESUMO

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is causally implicated in the development of lymphoid and epithelial tumors. Entry of virus requires fusion of virus envelopes and cell membranes. Fusion with B lymphocytes requires virus glycoprotein gB and a 3-part complex of glycoproteins, gHgLgp42. It is triggered by interactions between glycoprotein 42 (gp42) and HLA class II. However, fusion with epithelial cells is impeded by gp42 and instead is triggered by interactions between an unknown epithelial protein and a 2-part complex of gHgL. We report here that gHgL binds with high affinity to epithelial cells and that affinity of binding is increased by 3 orders of magnitude in the presence of Mn(2+). Binding and infection can be reduced by fibronectin and vitronectin, by down-regulation of integrin alphav, or by a peptide corresponding to 13 aa of gH which include a KGDE motif. Fusion of cells expressing gB and gHgL can be blocked by vitronectin or triggered by addition of soluble truncated integrins alphavbeta6 and alphavbeta8. We conclude that the direct interaction between EBV gHgL and integrins alphavbeta6 and alphavbeta8 can provide the trigger for fusion of EBV with an epithelial cell.


Assuntos
Células Epiteliais/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Integrinas/metabolismo , Manganês/farmacologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/virologia , Western Blotting , Células Epiteliais/virologia , Fibronectinas/farmacologia , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunoprecipitação , Integrinas/genética , Interferência de RNA , Vitronectina/farmacologia
17.
Elife ; 112022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35384838

RESUMO

Alveolar formation requires coordinated movement and interaction between alveolar epithelial cells, mesenchymal myofibroblasts, and endothelial cells/pericytes to produce secondary septa. These processes rely on the acquisition of distinct cellular properties to enable ligand secretion for cell-cell signaling and initiate morphogenesis through cellular contraction, cell migration, and cell shape change. In this study, we showed that mitochondrial activity and distribution play a key role in bestowing cellular functions on both alveolar epithelial cells and mesenchymal myofibroblasts for generating secondary septa to form alveoli in mice. These results suggest that mitochondrial function is tightly regulated to empower cellular machineries in a spatially specific manner. Indeed, such regulation via mitochondria is required for secretion of ligands, such as platelet-derived growth factor, from alveolar epithelial cells to influence myofibroblast proliferation and contraction/migration. Moreover, mitochondrial function enables myofibroblast contraction/migration during alveolar formation. Together, these findings yield novel mechanistic insights into how mitochondria regulate pivotal steps of alveologenesis. They highlight selective utilization of energy in cells and diverse energy demands in different cellular processes during development. Our work serves as a paradigm for studying how mitochondria control tissue patterning.


The lungs display an intricate, tree-shaped structure which enables the complex gas exchanges required for life. The end of each tiny 'branch' hosts delicate air sacs, or alveoli, which are further divided by internal walls called septa. In mammals, this final structure is acquired during the last stage of lung development. Then, many different types of cells in the immature alveoli multiply and reach the right location to start constructing additional septa. While the structural changes underlining alveoli maturation are well-studied, the energy requirements for that process remain poorly understood. In particular, the exact role of the mitochondria, the cellular compartments that power most life processes, is still unclear. Zhang et al. therefore set out to map, in detail, the role of mitochondria in alveolar development. Microscope imaging revealed how mitochondria were unevenly distributed within the lung cells of newborn mice. Mitochondria accumulated around the machinery that controls protein secretion in the epithelial cells that line the air sacs, and around the contractile apparatus in the underlying cells (the 'myofibroblasts'). Genetically altering the mice to reduce mitochondrial activity or perturb mitochondrial location in these two cell types produced defective alveoli with fewer septa, but it had no effect on lung development before alveoli formation. This suggests that the formation of alveoli requires more energy than other steps of lung development. Disrupting mitochondrial activity or location also compromised how epithelial cells produced chemical signals necessary for the contraction or migration of the myofibroblasts. Together, these results highlight the importance of tightly regulating mitochondrial activity and location during lung patterning. In the future, this insight could lay the groundwork to determine how energy requirements in various tissues shape other biological processes in health and disease.


Assuntos
Células Endoteliais , Alvéolos Pulmonares , Animais , Movimento Celular , Células Endoteliais/metabolismo , Pulmão/metabolismo , Camundongos , Mitocôndrias , Miofibroblastos/fisiologia , Alvéolos Pulmonares/metabolismo
18.
Front Immunol ; 13: 880887, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35634278

RESUMO

Macrophages are paracrine signalers that regulate tissular responses to injury through interactions with parenchymal cells. Connexin hemichannels have recently been shown to mediate efflux of ATP by macrophages, with resulting cytosolic calcium responses in adjacent cells. Here we report that lung macrophages with deletion of connexin 43 (MacΔCx43) had decreased ATP efflux into the extracellular space and induced a decreased cytosolic calcium response in co-cultured fibroblasts compared to WT macrophages. Furthermore, MacΔCx43 mice had decreased lung fibrosis after bleomycin-induced injury. Interrogating single cell data for human and mouse, we found that P2rx4 was the most highly expressed ATP receptor and calcium channel in lung fibroblasts and that its expression was increased in the setting of fibrosis. Fibroblast-specific deletion of P2rx4 in mice decreased lung fibrosis and collagen expression in lung fibroblasts in the bleomycin model. Taken together, these studies reveal a Cx43-dependent profibrotic effect of lung macrophages and support development of fibroblast P2rx4 as a therapeutic target for lung fibrosis.


Assuntos
Conexina 43 , Fibrose Pulmonar Idiopática , Trifosfato de Adenosina/metabolismo , Animais , Bleomicina/farmacologia , Cálcio/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout
19.
J Biol Chem ; 285(32): 24695-706, 2010 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-20519498

RESUMO

Integrin alphavbeta8 is a critical regulator of transforming growth factor beta activation in vasculogenesis during development, immune regulation, and endothelial/epithelial-mesenchymal homeostasis. Recent studies have suggested roles for integrin beta8 in the pathogenesis of chronic obstructive pulmonary disease, brain arteriovenous malformations, and select cancers (Araya, J., Cambier, S., Markovics, J. A., Wolters, P., Jablons, D., Hill, A., Finkbeiner, W., Jones, K., Broaddus, V. C., Sheppard, D., Barzcak, A., Xiao, Y., Erle, D. J., and Nishimura, S. L. (2007) J. Clin. Invest. 117, 3551-3562; Su, H., Kim, H., Pawlikowska, L., Kitamura, H., Shen, F., Cambier, S., Markovics, J., Lawton, M. T., Sidney, S., Bollen, A. W., Kwok, P. Y., Reichardt, L., Young, W. L., Yang, G. Y., and Nishimura, S. L. (2010) Am. J. Pathol. 176, 1018-1027; Culhane, A. C., and Quackenbush, J. (2009) Cancer Res. 69, 7480-7485; Cambier, S., Mu, D. Z., O'Connell, D., Boylen, K., Travis, W., Liu, W. H., Broaddus, V. C., and Nishimura, S. L. (2000) Cancer Res. 60, 7084-7093). Here we report the first identification and characterization of the promoter for ITGB8. We show that a SP binding site and a cyclic AMP response element (CRE) in the ITGB8 core promoter are required for its expression and that Sp1, Sp3, and several AP-1 transcription factors form a complex that binds to these sites in a p38-dependent manner. Furthermore, we demonstrate the requirement for Sp3, ATF-2, and p38 for the transcription and protein expression of integrin beta8. Additionally, reduction of SP3 or inhibition of p38 blocks alphavbeta8-mediated transforming growth factor beta activation. These results place integrin beta8 expression and activity under the control of ubiquitous transcription factors in a stress-activated and pro-inflammatory pathway.


Assuntos
Regulação Neoplásica da Expressão Gênica , Cadeias beta de Integrinas/metabolismo , Fator de Transcrição Sp3/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Ilhas de CpG , Células HeLa , Humanos , Inflamação , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
20.
Am J Physiol Lung Cell Mol Physiol ; 300(3): L391-401, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21224216

RESUMO

Reepithelialization of remodeled air spaces with bronchial epithelial cells is a prominent pathological finding in idiopathic pulmonary fibrosis (IPF) and is implicated in IPF pathogenesis. Recent studies suggest that epithelial senescence is a risk factor for development of IPF, indicating such reepithelialization may be influenced by the acceleration of cellular senescence. Among the sirtuin (SIRT) family, SIRT6, a class III histone deacetylase, has been demonstrated to antagonize senescence. We evaluated the senescence of bronchiolization in association with SIRT6 expression in IPF lung. Senescence-associated ß-galactosidase staining and immunohistochemical detection of p21 were performed to evaluate cellular senescence. As a model for transforming growth factor (TGF)-ß-induced senescence of abnormal reepithelialization, we used primary human bronchial epithelial cells (HBEC). The changes of SIRT6, p21, and interleukin (IL)-1ß expression levels in HBEC, as well as type I collagen expression levels in fibroblasts, were evaluated. In IPF lung samples, an increase in markers of senescence and SIRT6 expression was found in the bronchial epithelial cells lining cystically remodeled air spaces. We found that TGF-ß induced senescence in primary HBEC by increasing p21 expression, and, whereas TGF-ß also induced SIRT6, it was not sufficient to inhibit cellular senescence. However, overexpression of SIRT6 efficiently inhibited TGF-ß-induced senescence via proteasomal degradation of p21. TGF-ß-induced senescent HBEC secreted increased amounts of IL-1ß, which was sufficient to induce myofibroblast differentiation in fibroblasts. These findings suggest that accelerated epithelial senescence plays a role in IPF pathogenesis through perpetuating abnormal epithelial-mesenchymal interactions, which can be antagonized by SIRT6.


Assuntos
Brônquios/patologia , Senescência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose Pulmonar Idiopática/patologia , Sirtuínas/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Diferenciação Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Interleucina-1beta/metabolismo , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , beta-Galactosidase/metabolismo
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