RESUMO
The evolution of T cell molecular signatures in the distal lung of patients with severe pneumonia is understudied. Here, we analyzed T cell subsets in longitudinal bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia, including unvaccinated patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or with respiratory failure not linked to pneumonia. In patients with SARS-CoV-2 pneumonia, activation of interferon signaling pathways, low activation of the NF-κB pathway and preferential targeting of spike and nucleocapsid proteins early after intubation were associated with favorable outcomes, whereas loss of interferon signaling, activation of NF-κB-driven programs and specificity for the ORF1ab complex late in disease were associated with mortality. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize individuals who recover, whereas responses against nonstructural proteins and activation of NF-κB are associated with poor outcomes.
RESUMO
Some patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe pneumonia and acute respiratory distress syndrome1 (ARDS). Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from that in other types of pneumonia2. Here we investigate SARS-CoV-2 pathobiology by characterizing the immune response in the alveoli of patients infected with the virus. We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens, and analysed them using flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA sequencing on 10 bronchoalveolar lavage fluid samples collected from patients with severe coronavirus disease 2019 (COVID-19) within 48 h of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-γ to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages containing SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Macrófagos Alveolares/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , SARS-CoV-2/patogenicidade , Linfócitos T/imunologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , COVID-19/genética , Estudos de Coortes , Humanos , Interferon gama/imunologia , Interferons/imunologia , Interferons/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Pneumonia Viral/genética , RNA-Seq , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Análise de Célula Única , Linfócitos T/metabolismo , Fatores de TempoRESUMO
Regulatory T cells (Treg cells), a distinct subset of CD4+ T cells, are necessary for the maintenance of immune self-tolerance and homeostasis1,2. Recent studies have demonstrated that Treg cells exhibit a unique metabolic profile, characterized by an increase in mitochondrial metabolism relative to other CD4+ effector subsets3,4. Furthermore, the Treg cell lineage-defining transcription factor, Foxp3, has been shown to promote respiration5,6; however, it remains unknown whether the mitochondrial respiratory chain is required for the T cell-suppression capacity, stability and survival of Treg cells. Here we report that Treg cell-specific ablation of mitochondrial respiratory chain complex III in mice results in the development of fatal inflammatory disease early in life, without affecting Treg cell number. Mice that lack mitochondrial complex III specifically in Treg cells displayed a loss of T cell-suppression capacity without altering Treg cell proliferation and survival. Treg cells deficient in complex III showed decreased expression of genes associated with Treg function, whereas Foxp3 expression remained stable. Loss of complex III in Treg cells increased DNA methylation as well as the metabolites 2-hydroxyglutarate (2-HG) and succinate that inhibit the ten-eleven translocation (TET) family of DNA demethylases7. Thus, Treg cells require mitochondrial complex III to maintain immune regulatory gene expression and suppressive function.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Tolerância a Antígenos Próprios/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Desmetilação do DNA , Metilação de DNA , Transporte de Elétrons , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Glutaratos/metabolismo , Inflamação/genética , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Tolerância a Antígenos Próprios/genética , Ácido Succínico/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/enzimologiaRESUMO
Pulmonary fibrosis is a relentlessly progressive and often fatal disease with a paucity of available therapies. Genetic evidence implicates disordered epithelial repair, which is normally achieved by the differentiation of small cuboidal alveolar type 2 (AT2) cells into large, flattened alveolar type 1 (AT1) cells as an initiating event in pulmonary fibrosis pathogenesis. Using models of pulmonary fibrosis in young adult and old mice and a model of adult alveologenesis after pneumonectomy, we show that administration of ISRIB, a small molecule that restores protein translation by EIF2B during activation of the integrated stress response (ISR), accelerated the differentiation of AT2 into AT1 cells. Accelerated epithelial repair reduced the recruitment of profibrotic monocyte-derived alveolar macrophages and ameliorated lung fibrosis. These findings suggest a dysfunctional role for the ISR in regeneration of the alveolar epithelium after injury with implications for therapy.
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Acetamidas/farmacologia , Células Epiteliais Alveolares/efeitos dos fármacos , Cicloexilaminas/farmacologia , Proteostase/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Acetamidas/uso terapêutico , Fatores Etários , Células Epiteliais Alveolares/citologia , Animais , Amianto , Bleomicina , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Cicloexilaminas/uso terapêutico , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteostase/fisiologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Estresse Fisiológico/efeitos dos fármacosRESUMO
BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is well characterized by type 2 (T2) inflammation characterized by eosinophilia in Western countries. However, the presence and roles of neutrophils in T2 CRSwNP are poorly understood. OBJECTIVE: We sought to clarify accumulation and inflammatory roles of neutrophils in CRSwNP in a Western population. METHODS: Sinonasal tissues and nasal lavage fluids were obtained from control patients and patients with CRS, and neutrophil markers were determined by ELISA. The presence of neutrophils in tissue was determined by flow cytometry. The gene expression profiles in neutrophils were determined by RNA sequencing. RESULTS: A neutrophil marker elastase was selectively elevated in nasal polyp (NP) tissue, whereas eosinophilic cationic protein (an eosinophil marker) was elevated in both uncinate and NP tissues of CRSwNP patients. Nasal lavage fluid myeloperoxidase (another neutrophil marker) was also significantly elevated in CRSwNP compared to control patients. Neutrophil markers were more greatly elevated in CRSwNP patients with recurrent disease. Flow cytometric analysis confirmed that neutrophil numbers were significantly elevated in NPs compared to control tissues. RNA sequencing analysis found that 344 genes were >3-fold and significantly elevated in NP neutrophils compared to peripheral blood neutrophils. Gene Ontology analysis suggested that the elevated genes in NP neutrophils were significantly associated with activation. Results suggest that neutrophils are accumulated in T2 NP tissues and that accumulated neutrophils are highly activated and contribute to inflammation in NPs. CONCLUSIONS: Neutrophils may play a heretofore unrecognized meaningful role in the pathogenesis of CRSwNP in Western countries and may be a potentially important therapeutic target in T2 CRSwNP.
Assuntos
Pólipos Nasais , Rinite , Sinusite , Biomarcadores , Doença Crônica , Humanos , Inflamação/patologia , Pólipos Nasais/patologia , Neutrófilos/patologia , Rinite/patologia , Sinusite/patologiaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 180 million people since the onset of the pandemic. Despite similar viral load and infectivity rates between children and adults, children rarely develop severe illness. Differences in the host response to the virus at the primary infection site are among the mechanisms proposed to account for this disparity. Our objective was to investigate the host response to SARS-CoV-2 in the nasal mucosa in children and adults and compare it with the host response to respiratory syncytial virus (RSV) and influenza virus. We analyzed clinical outcomes and gene expression in the nasal mucosa of 36 children with SARS-CoV-2, 24 children with RSV, 9 children with influenza virus, 16 adults with SARS-CoV-2, and 7 healthy pediatric and 13 healthy adult controls. In both children and adults, infection with SARS-CoV-2 led to an IFN response in the nasal mucosa. The magnitude of the IFN response correlated with the abundance of viral reads, not the severity of illness, and was comparable between children and adults infected with SARS-CoV-2 and children with severe RSV infection. Expression of ACE2 and TMPRSS2 did not correlate with age or presence of viral infection. SARS-CoV-2-infected adults had increased expression of genes involved in neutrophil activation and T-cell receptor signaling pathways compared with SARS-CoV-2-infected children, despite similar severity of illness and viral reads. Age-related differences in the immune response to SARS-CoV-2 may place adults at increased risk of developing severe illness.
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Envelhecimento/imunologia , COVID-19/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade nas Mucosas , Mucosa Nasal/imunologia , SARS-CoV-2/imunologia , Adolescente , Fatores Etários , Enzima de Conversão de Angiotensina 2/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Mucosa Nasal/virologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Serina Endopeptidases/imunologiaRESUMO
In inflamed human tissues, we often find intact eosinophilic granules, but not eosinophils themselves. Eosinophils, tissue-dwelling granulocytes with several homeostatic roles, have a surprising association with fibrinogen and tissue remodeling. Fibrinogen is a complex glycoprotein with regulatory roles in hemostasis, tumor development, wound healing, and atherogenesis. Despite its significance, the functional link between eosinophils and fibrinogen is not understood. We tested IL-5-primed mouse bone marrow-derived and human blood-sorted eosinophil activity against FITC-linked fibrinogen substrates. The interactions between these scaffolds and adhering eosinophils were quantified using three-dimensional laser spectral, confocal, and transmission electron microscopy. Eosinophils were labeled with major basic protein (MBP) Ab to visualize granules and assessed by flow cytometry. Both mouse and human eosinophils showed firm adhesion and degraded up to 27 ± 3.1% of the substrate area. This co-occurred with active MBP-positive granule release and the expression of integrin CD11b. Mass spectrometry analysis of fibrinogen proteolytic reactions detected the presence of eosinophil peroxidase, MBP, and fibrin α-, ß-, and γ-chains. Eosinophil activity was adhesion dependent, as a blocking Ab against CD11b significantly reduced adhesion, degranulation, and fibrinogenolysis. Although adhered, eosinophils exhibited no proteolytic activity on collagen matrices. Cytolytic degranulation was defined by loss of membrane integrity, cell death, and presence of cell-free granules. From transmission electron microscopy images, we observed only fibrinogen-exposed eosinophils undergoing this process. To our knowledge, this is the first report to show that fibrinogen is a specific trigger for cytolytic eosinophil degranulation with implications in human disease.
Assuntos
Eosinófilos/imunologia , Fibrinogênio/metabolismo , Inflamação/metabolismo , Animais , Antígeno CD11b/metabolismo , Adesão Celular , Morte Celular , Degranulação Celular , Células Cultivadas , Citotoxicidade Imunológica , Proteína Básica Maior de Eosinófilos/metabolismo , Humanos , Inflamação/imunologia , Interleucina-5/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Vesículas Secretórias/metabolismoRESUMO
BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), and an intolerance of medications that inhibit cyclooxygenase-1. Patients with AERD have more severe upper and lower respiratory tract disease than do aspirin-tolerant patients with CRSwNP. A dysregulation in arachidonic acid metabolism is thought to contribute to the enhanced sinonasal inflammation in AERD. OBJECTIVE: Our aim was to utilize an unbiased approach investigating arachidonic acid metabolic pathways in AERD. METHODS: Single-cell RNA sequencing (10× Genomics, Pleasanton, Calif) was utilized to compare the transcriptional profile of nasal polyp (NP) cells from patients with AERD and patients with CRSwNP and map differences in the expression of select genes among identified cell types. Findings were confirmed by traditional real-time PCR. Lipid mediators in sinonasal tissue were measured by mass spectrometry. Localization of various proteins within NPs was assessed by immunofluorescence. RESULTS: The gene encoding for 15-lipooxygenase (15-LO), ALOX15, was significantly elevated in NPs of patients with AERD compared to NPs of patients with CRSwNP (P < .05) or controls (P < .001). ALOX15 was predominantly expressed by epithelial cells. Expression levels significantly correlated with radiographic sinus disease severity (r = 0.56; P < .001) and were associated with asthma. The level of 15-oxo-eicosatetraenoic acid (15-Oxo-ETE), a downstream product of 15-LO, was significantly elevated in NPs from patients with CRSwNP (27.93 pg/mg of tissue) and NPs from patients with AERD (61.03 pg/mg of tissue) compared to inferior turbinate tissue from controls (7.17 pg/mg of tissue [P < .001]). Hydroxyprostaglandin dehydrogenase, an enzyme required for 15-Oxo-ETE synthesis, was predominantly expressed in mast cells and localized near 15-LO+ epithelium in NPs from patients with AERD. CONCLUSIONS: Epithelial and mast cell interactions, leading to the synthesis of 15-Oxo-ETE, may contribute to the dysregulation of arachidonic acid metabolism via the 15-LO pathway and to the enhanced sinonasal disease severity observed in AERD.
Assuntos
Araquidonato 15-Lipoxigenase/imunologia , Asma Induzida por Aspirina/imunologia , Transtornos Respiratórios/imunologia , Adulto , Araquidonato 15-Lipoxigenase/metabolismo , Asma Induzida por Aspirina/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Respiratórios/metabolismoRESUMO
Concomitant dramatic increase in prevalence of allergic and metabolic diseases is part of a modern epidemic afflicting technologically advanced societies. While clinical evidence points to clear associations between various metabolic factors and atopic disease, there is still a very limited understanding of the mechanisms that link the two. Dysregulation of central metabolism in metabolic syndrome, obesity, diabetes, and dyslipidemia has a systemic impact on multiple tissues and organs, including cells of the epithelial barrier. While much of epithelial research in allergy has focused on the immune-driven processes, a growing number of recent studies have begun to elucidate the role of metabolic components of disease. This review will revisit clinical evidence for the relationship between metabolic and allergic diseases, as well as discuss potential mechanisms driving metabolic dysfunction of the epithelial barrier. Among them, novel studies highlight links between dysregulation of the insulin pathway, glucose metabolism, and loss of epithelial differentiation in asthma. Studies of mitochondrial structure and bioenergetics in lean and obese asthmatic phenotypes recently came to light to provide a novel framework linking changes in tricarboxylic acid cycle and oxidative phosphorylation with arginine metabolism and nitric oxide bioavailability. New research established connections between arachidonate metabolism, autophagy, and airway disease, as well as systemic dyslipidemia in atopic dermatitis and ceramide changes in the epidermis. Taken together, studies of metabolism have a great potential to open doors to a new class of therapeutic strategies, better characterization of disease endotypes, as well as enable a systems biology approach to mechanisms of allergic disease.
Assuntos
Suscetibilidade a Doenças , Metabolismo Energético , Células Epiteliais/metabolismo , Homeostase , Hipersensibilidade/etiologia , Hipersensibilidade/metabolismo , Animais , Biomarcadores , Diabetes Mellitus/metabolismo , Humanos , Resistência à Insulina , Redes e Vias Metabólicas , Mitocôndrias , Obesidade/complicações , Obesidade/etiologia , Obesidade/metabolismo , Transdução de SinaisRESUMO
Pulmonary hypertension (PH) and right ventricular (RV) hypertrophy frequently develop in patients with hypoxic lung disease. Chronic alveolar hypoxia (CH) promotes sustained pulmonary vasoconstriction and pulmonary artery (PA) remodeling by acting on lung cells, resulting in the development of PH. RV hypertrophy develops in response to PH, but coronary arterial hypoxemia in CH may influence that response by activating HIF-1α (hypoxia-inducible factor 1α) and/or HIF-2α in cardiomyocytes. Indeed, other studies show that the attenuation of PH in CH fails to prevent RV remodeling, suggesting that PH-independent factors regulate RV hypertrophy. Therefore, we examined the role of HIFs in RV remodeling in CH-induced PH. We deleted HIF-1α and/or HIF-2α in hearts of adult mice that were then housed under normoxia or CH (10% O2) for 4 weeks. RNA-sequencing analysis of the RV revealed that HIF-1α and HIF-2α regulate the transcription of largely distinct gene sets during CH. RV systolic pressure increased, and RV hypertrophy developed in CH. The deletion of HIF-1α in smooth muscle attenuated the CH-induced increases in RV systolic pressure but did not decrease hypertrophy. The deletion of HIF-1α in cardiomyocytes amplified RV remodeling; this was abrogated by the simultaneous loss of HIF-2α. CH decreased stroke volume and cardiac output in wild-type but not in HIF-1α-deficient hearts, suggesting that CH may cause cardiac dysfunction via HIF-dependent signaling. Collectively, these data reveal that HIF-1 and HIF-2 act together in RV cardiomyocytes to orchestrate RV remodeling in CH, with HIF-1 playing a protective role rather than driving hypertrophy.
Assuntos
Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/complicações , Função Ventricular Direita/fisiologia , Remodelação Ventricular/fisiologia , Animais , Doença Crônica , Deleção de Genes , Regulação da Expressão Gênica , Ontologia Genética , Hipertensão Pulmonar/genética , Integrases/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Transcrição Gênica , Função Ventricular Direita/genética , Remodelação Ventricular/genéticaRESUMO
Rationale: The identification of informative elements of the host response to infection may improve the diagnosis and management of bacterial pneumonia. Objectives: To determine whether the absence of alveolar neutrophilia can exclude bacterial pneumonia in critically ill patients with suspected infection and to test whether signatures of bacterial pneumonia can be identified in the alveolar macrophage transcriptome. Methods: We determined the test characteristics of alveolar neutrophilia for the diagnosis of bacterial pneumonia in three cohorts of mechanically ventilated patients. In one cohort, we also isolated macrophages from alveolar lavage fluid and used the transcriptome to identify signatures of bacterial pneumonia. Finally, we developed a humanized mouse model of Pseudomonas aeruginosa pneumonia to determine if pathogen-specific signatures can be identified in human alveolar macrophages. Measurements and Main Results: An alveolar neutrophil percentage less than 50% had a negative predictive value of greater than 90% for bacterial pneumonia in both the retrospective (n = 851) and validation cohorts (n = 76 and n = 79). A transcriptional signature of bacterial pneumonia was present in both resident and recruited macrophages. Gene signatures from both cell types identified patients with bacterial pneumonia with test characteristics similar to alveolar neutrophilia. Conclusions: The absence of alveolar neutrophilia has a high negative predictive value for bacterial pneumonia in critically ill patients with suspected infection. Macrophages can be isolated from alveolar lavage fluid obtained during routine care and used for RNA-Seq analysis. This novel approach may facilitate a longitudinal and multidimensional assessment of the host response to bacterial pneumonia.
Assuntos
Antibacterianos/uso terapêutico , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Pneumonia Bacteriana/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Respiração Artificial , Idoso , Animais , Estudos de Coortes , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.
Assuntos
Células Cultivadas/patologia , Células Epiteliais/patologia , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Análise de Sequência de RNA , Células-Tronco/patologia , Transcriptoma , Animais , Modelos Animais de Doenças , Feminino , Humanos , MasculinoRESUMO
Pediatric acute lung injury, usually because of pneumonia, has a mortality rate of more than 20% and an incidence that rivals that of all childhood cancers combined. CD4+ T-cells coordinate the immune response to pneumonia but fail to function robustly among the very young, who have poor outcomes from lung infection. We hypothesized that DNA methylation represses a mature CD4+ T-cell transcriptional program in neonates with pneumonia. Here, we found that neonatal mice (3-4 days old) aspirated with Escherichia coli bacteria had a higher mortality rate than juvenile mice (11-14 days old). Transcriptional profiling with an unsupervised RNA-Seq approach revealed that neonates displayed an attenuated lung CD4+ T-cell transcriptional response to pneumonia compared with juveniles. Unlike neonates, juveniles up-regulated a robust set of canonical T-cell immune response genes. DNA methylation profiling with modified reduced representation bisulfite sequencing revealed 44,119 differentially methylated CpGs, which preferentially clustered around transcriptional start sites and CpG islands. A methylation difference-filtering algorithm detected genes with a high likelihood of differential promoter methylation regulating their expression; these 731 loci encoded important immune response and tissue-protective T-cell pathway components. Disruption of DNA methylation with the hypomethylating agent decitabine induced plasticity in the lung CD4+ T-cell marker phenotype. Altogether, multidimensional profiling suggested that DNA methylation within the promoters of a core set of CD4+ T-cell pathway genes contributes to the hyporesponsive neonatal immune response to pneumonia. These findings also suggest that DNA methylation could serve as a mechanistic target for disease-modifying therapies in pediatric lung infection and injury.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Metilação de DNA , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Pneumonia/imunologia , Animais , Animais Recém-Nascidos , Linfócitos T CD4-Positivos/metabolismo , Ilhas de CpG , Epigênese Genética , Infecções por Escherichia coli/genética , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/genética , Ativação TranscricionalRESUMO
Idiopathic pulmonary fibrosis (IPF) is characterized by progressive scarring of the lung parenchyma, leading to respiratory failure and death. High resolution computed tomography of the chest is often diagnostic for IPF, but its cost and the risk of radiation exposure limit its use as a screening tool even in patients at high risk for the disease. In patients with lung cancer, investigators have detected transcriptional signatures of disease in airway and nasal epithelial cells distal to the site of disease that are clinically useful as screening tools. Here we assessed the feasibility of distinguishing patients with IPF from age-matched controls through transcriptomic profiling of nasal epithelial curettage samples, which can be safely and repeatedly sampled over the course of a patient's illness. We recruited 10 patients with IPF and 23 age-matched healthy control subjects. Using 3' messenger RNA sequencing (mRNA-seq), we identified 224 differentially expressed genes, most of which were upregulated in patients with IPF compared with controls. Pathway enrichment analysis revealed upregulation of pathways related to immune response and inflammatory signaling in IPF patients compared with controls. These findings support the concept that fibrosis is associated with upregulation of inflammatory pathways across the respiratory epithelium with possible implications for disease detection and pathobiology.
Assuntos
Fibrose Pulmonar Idiopática/metabolismo , Mediadores da Inflamação/metabolismo , Mucosa Nasal/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , Idoso , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/patologiaRESUMO
Mast cells are critical in the pathogenesis of allergic disease due to the release of preformed and newly synthesized mediators, yet the mechanisms controlling mast cell activation are not well understood. Members of the tetraspanin family are recently emerging as modulators of FcεRI-mediated mast cell activation; however, mechanistic understanding of their function is currently lacking. The tetraspanin CD151 is a poorly understood member of this family and is specifically induced on mouse and human mast cells upon FcεRI aggregation but its functional effects are unknown. In this study, we show that CD151 deficiency significantly exacerbates the IgE-mediated late phase inflammation in a murine model of passive cutaneous anaphylaxis. Ex vivo, FcεRI stimulation of bone marrow-derived mast cells from CD151(-/-) mice resulted in significantly enhanced expression of proinflammatory cytokines IL-4, IL-13, and TNF-α compared with wild-type controls. However, FcεRI-induced mast cell degranulation was unaffected. At the molecular signaling level, CD151 selectively regulated IgE-induced activation of ERK1/2 and PI3K, associated with cytokine production, but had no effect on the phospholipase Cγ1 signaling, associated with degranulation. Collectively, our data indicate that CD151 exerts negative regulation over IgE-induced late phase responses and cytokine production in mast cells.
Assuntos
Imunomodulação , Mastócitos/imunologia , Mastócitos/metabolismo , Receptores de IgE/metabolismo , Tetraspanina 24/metabolismo , Anafilaxia/genética , Anafilaxia/imunologia , Anafilaxia/metabolismo , Animais , Degranulação Celular/genética , Degranulação Celular/imunologia , Diferenciação Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Hipersensibilidade/diagnóstico , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Imunoglobulina E/imunologia , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases , Mastócitos/citologia , Camundongos , Camundongos Knockout , Anafilaxia Cutânea Passiva , Fosfatidilinositol 3-Quinases , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Tetraspanina 24/deficiência , Tetraspanina 24/genéticaRESUMO
γ-Tocopherol increases responses to allergen challenge in allergic adult mice, but it is not known whether γ-tocopherol regulates the development of allergic disease. Development of allergic disease often occurs early in life. In clinical studies and animal models, offspring of allergic mothers have increased responsiveness to allergen challenge. Therefore, we determined whether γ-tocopherol augments development of allergic responses in offspring of allergic female mice. Allergic female mice were supplemented with γ-tocopherol starting at mating. The pups from allergic mothers developed allergic lung responses, whereas pups from saline-treated mothers did not respond to allergen challenge. The γ-tocopherol supplementation of allergic female mice increased the numbers of eosinophils twofold in the pup bronchoalveolar lavage and lungs after allergen challenge. There was also about a twofold increase in pup lung CD11b(+) subsets of CD11c(+) dendritic cells and in numbers of these dendritic cells expressing the transcription factor IRF4. There was no change in several CD11b(-) dendritic cell subsets. Furthermore, maternal supplementation with γ-tocopherol increased the number of fetal liver CD11b(+)CD11c(+) dendritic cells twofold in utero. In the pups, γ-tocopherol increased lung expression of the inflammatory mediators CCL11, amphiregulin, activin A, and IL-5. In conclusion, maternal supplementation with γ-tocopherol increased fetal development of subsets of dendritic cells that are critical for allergic responses and increased development of allergic responses in pups from allergic mothers. These results have implications for supplementation of allergic mothers with γ-tocopherol in prenatal vitamins.
Assuntos
Asma/imunologia , Células Dendríticas/imunologia , Suplementos Nutricionais/efeitos adversos , Pneumonia/imunologia , gama-Tocoferol/efeitos adversos , Animais , Asma/induzido quimicamente , Antígenos CD11/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Exposição Materna , Troca Materno-Fetal , Camundongos Endogâmicos C57BL , Pneumonia/induzido quimicamente , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/imunologia , gama-Tocoferol/administração & dosagemRESUMO
Prospective epidemiological studies, observational cross-sectional studies and some randomised prevention trials have demonstrated inconsistent findings of the impact of vitamin E on asthma risk. The goals of this study were to explore whether this differing association of vitamin E on asthma risk is due to an interaction of vitamin E isoforms. To address this question, in a population-based asthma incidence study we assessed the interaction between the plasma concentrations of vitamin E isoforms α-tocopherol and γ-tocopherol on asthma risk. Second, to understand the mechanisms of any interaction of these isoforms, we conducted experimental supplementation of α-tocopherol and γ-tocopherol isoforms in mice on the outcome of allergic airway inflammation. We found that in the highest γ-tocopherol tertile, low levels of α-tocopherol were associated with increased asthma risk, while highest tertile α-tocopherol levels trended to be protective. Similarly, in a mouse model of asthma, diet supplementation with α-tocopherol decreased lung inflammation in response to house dust mite (HDM) challenge. In contrast, diet supplementation with γ-tocopherol increased lung inflammation in response to HDM. These human and animal studies provide evidence for the competing effects of the vitamin E isoforms, in physiological concentrations, on asthma and allergic airway disease.
Assuntos
Asma/sangue , alfa-Tocoferol/sangue , gama-Tocoferol/sangue , Animais , Humanos , Camundongos , Isoformas de Proteínas/sangue , Hipersensibilidade Respiratória/sangueRESUMO
Tissue transglutaminase 2 (TG2) is an enzyme with multiple functions, including catalysis of serotonin conjugation to proteins (serotonylation). Previous research indicates that TG2 expression is upregulated in human asthma and in the lung endothelium of ovalbumin (OVA)-challenged mice. It is not known whether endothelial cell TG2 is required for allergic inflammation. Therefore, to determine whether endothelial cell TG2 regulates allergic inflammation, mice with an endothelial cell-specific deletion of TG2 were generated, and these mice were sensitized and challenged in the airways with OVA. Deletion of TG2 in endothelial cells blocked OVA-induced serotonylation in lung endothelial cells, but not lung epithelial cells. Interestingly, deletion of endothelial TG2 reduced allergen-induced increases in respiratory system resistance, number of eosinophils in the bronchoalveolar lavage, and number of eosinophils in the lung tissue. Endothelial cell deletion of TG2 did not alter expression of adhesion molecules, cytokines, or chemokines that regulate leukocyte recruitment, consistent with other studies, demonstrating that deletion of endothelial cell signals does not alter lung cytokines and chemokines during allergic inflammation. Taken together, the data indicate that endothelial cell TG2 is required for allergic inflammation by regulating the recruitment of eosinophils into OVA-challenged lungs. In summary, TG2 functions as a critical signal for allergic lung responses. These data identify potential novel targets for intervention in allergy/asthma.
Assuntos
Asma/enzimologia , Células Endoteliais/enzimologia , Proteínas de Ligação ao GTP/fisiologia , Pulmão/enzimologia , Transglutaminases/fisiologia , Animais , Asma/patologia , Quimiocinas/metabolismo , Eosinófilos/imunologia , Feminino , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína 2 Glutamina gama-GlutamiltransferaseRESUMO
α-Tocopherol blocks responses to allergen challenge in allergic adult mice, but it is not known whether α-tocopherol regulates the development of allergic disease. Development of allergic disease often occurs early in life. In clinical studies and animal models, offspring of allergic mothers have increased responsiveness to allergen challenge. Therefore, we determined whether α-tocopherol blocked development of allergic responses in offspring of allergic female mice. Allergic female mice were supplemented with α-tocopherol starting at mating. The pups from allergic mothers developed allergic lung responses, whereas pups from saline-treated mothers did not respond to the allergen challenge, and α-tocopherol supplementation of allergic female mice resulted in a dose-dependent reduction in eosinophils in the pup bronchoalveolar lavage and lungs after allergen challenge. There was also a reduction in pup lung CD11b(+) dendritic cell subsets that are critical to development of allergic responses, but there was no change in several CD11b(-) dendritic cell subsets. Furthermore, maternal supplementation with α-tocopherol reduced the number of fetal liver CD11b(+) dendritic cells in utero. In the pups, there was reduced allergen-induced lung mRNA expression of IL-4, IL-33, TSLP, CCL11, and CCL24. Cross-fostering pups at the time of birth demonstrated that α-tocopherol had a regulatory function in utero. In conclusion, maternal supplementation with α-tocopherol reduced fetal development of subsets of dendritic cells that are critical for allergic responses and reduced development of allergic responses in pups from allergic mothers. These results have implications for supplementation of allergic mothers with α-tocopherol.