RESUMO
The epidermal growth factor receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis after injury; however, the mechanism by which Amphiregulin contributes to wound repair remains unknown. Here we show that Amphiregulin functioned by releasing bioactive transforming growth factor beta (TGF-ß) from latent complexes via integrin-αV activation. Using acute injury models in two different tissues, we found that by inducing TGF-ß activation on mesenchymal stromal cells (pericytes), Amphiregulin induced their differentiation into myofibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration after acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.
Assuntos
Anfirregulina/metabolismo , Macrófagos/metabolismo , Pericitos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular/fisiologia , Feminino , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismoRESUMO
BACKGROUND: Cancer-associated fibroblasts (CAFs) are a dominant cell type in the stroma of non-small cell lung cancer (NSCLC). Fibroblast heterogeneity reflects subpopulations of CAFs, which can influence prognosis and treatment efficacy. We describe the subtypes of CAFs in NSCLC. METHODS: Primary human NSCLC resections were assessed by flow cytometry and multiplex immunofluorescence for markers of fibroblast activation which allowed identification of CAF subsets. Survival data were analysed for our NSCLC cohort consisting of 163 patients to understand prognostic significance of CAF subsets. RESULTS: We identified five CAF populations, termed CAF S1-S5. CAF-S5 represents a previously undescribed population, and express FAP and PDPN but lack the myofibroblast marker αSMA, whereas CAF-S1 populations express all three. CAF-S5 are spatially further from tumour regions then CAF-S1 and scRNA data demonstrate an inflammatory phenotype. The presence of CAF-S1 or CAF-S5 is correlated to worse survival outcome in NSCLC, despite curative resection, highlighting the prognostic importance of CAF subtypes in NSCLC. TCGA data suggest the predominance of CAF-S5 has a poor prognosis across several cancer types. CONCLUSION: This study describes the fibroblast heterogeneity in NSCLC and the prognostic importance of the novel CAF-S5 subset where its presence correlates to worse survival outcome.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Glicoproteínas de Membrana , Proteínas de Membrana , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Prognóstico , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Feminino , Masculino , Endopeptidases , Gelatinases/metabolismo , Gelatinases/genética , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Idoso , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Microambiente TumoralRESUMO
Immunopathology occurs in the lung and spleen in fatal coronavirus disease (COVID-19), involving monocytes/macrophages and plasma cells. Antiinflammatory therapy reduces mortality, but additional therapeutic targets are required. We aimed to gain mechanistic insight into COVID-19 immunopathology by targeted proteomic analysis of pulmonary and splenic tissues. Lung parenchymal and splenic tissue was obtained from 13 postmortem examinations of patients with fatal COVID-19. Control tissue was obtained from cancer resection samples (lung) and deceased organ donors (spleen). Protein was extracted from tissue by phenol extraction. Olink multiplex immunoassay panels were used for protein detection and quantification. Proteins with increased abundance in the lung included MCP-3, antiviral TRIM21, and prothrombotic TYMP. OSM and EN-RAGE/S100A12 abundance was correlated and associated with inflammation severity. Unsupervised clustering identified "early viral" and "late inflammatory" clusters with distinct protein abundance profiles, and differences in illness duration before death and presence of viral RNA. In the spleen, lymphocyte chemotactic factors and CD8A were decreased in abundance, and proapoptotic factors were increased. B-cell receptor signaling pathway components and macrophage colony stimulating factor (CSF-1) were also increased. Additional evidence for a subset of host factors (including DDX58, OSM, TYMP, IL-18, MCP-3, and CSF-1) was provided by overlap between 1) differential abundance in spleen and lung tissue; 2) meta-analysis of existing datasets; and 3) plasma proteomic data. This proteomic analysis of lung parenchymal and splenic tissue from fatal COVID-19 provides mechanistic insight into tissue antiviral responses, inflammation and disease stages, macrophage involvement, pulmonary thrombosis, splenic B-cell activation, and lymphocyte depletion.
Assuntos
COVID-19/imunologia , Regulação da Expressão Gênica/imunologia , Pulmão/imunologia , SARS-CoV-2/imunologia , Baço/imunologia , Idoso , Idoso de 80 Anos ou mais , Autopsia , Feminino , Humanos , Inflamação/imunologia , Masculino , ProteômicaRESUMO
Rationale: In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown.Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury.Methods: Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by in situ viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence.Measurements and Main Results: Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues.Conclusions: Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.
Assuntos
COVID-19/imunologia , Inflamação/virologia , Pulmão/imunologia , Insuficiência de Múltiplos Órgãos/virologia , SARS-CoV-2/imunologia , Idoso , Idoso de 80 Anos ou mais , Autopsia , Biópsia , COVID-19/patologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Feminino , Imunofluorescência , Humanos , Inflamação/imunologia , Inflamação/patologia , Pulmão/patologia , Pulmão/virologia , Masculino , Insuficiência de Múltiplos Órgãos/imunologia , Insuficiência de Múltiplos Órgãos/patologia , SARS-CoV-2/patogenicidade , Índice de Gravidade de DoençaRESUMO
Eosinophils are key effector cells in allergic diseases. Here we investigated Mcl-1 (an anti-apoptotic protein) in experimental allergic airway inflammation using transgenic overexpressing human Mcl-1 mice (hMcl-1) and reducing Mcl-1 by a cyclin-dependent kinase inhibitor. Overexpression of Mcl-1 exacerbated allergic airway inflammation, with increased bronchoalveolar lavage fluid cellularity, eosinophil numbers and total protein, and an increase in airway mucus production. Eosinophil apoptosis was suppressed by Mcl-1 overexpression, with this resistance to apoptosis attenuated by cyclin-dependent kinase inhibition which also rescued Mcl-1-exacerbated allergic airway inflammation. We propose that targeting Mcl-1 may be beneficial in treatment of allergic airway disease.
Assuntos
Asma/genética , Eosinófilos/patologia , Regulação da Expressão Gênica , Hipersensibilidade/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , RNA/genética , Animais , Apoptose , Asma/metabolismo , Asma/patologia , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Eosinófilos/metabolismo , Feminino , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Contagem de Leucócitos , Camundongos , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossínteseRESUMO
Acute respiratory distress syndrome (ARDS) is the often fatal sequelae of a broad range of precipitating conditions. Despite decades of intensive research and clinical trials there remain no therapies in routine clinical practice that target the dysregulated and overwhelming inflammatory response that characterises ARDS. Neutrophils play a central role in the initiation, propagation and resolution of this complex inflammatory environment by migrating into the lung and executing a variety of pro-inflammatory functions. These include degranulation with liberation of bactericidal proteins, release of cytokines and reactive oxygen species as well as production of neutrophil extracellular traps. Although these functions are advantageous in clearing bacterial infection, the consequence of associated tissue damage, the contribution to worsening acute inflammation and prolonged neutrophil lifespan at sites of inflammation are deleterious. In this review, the importance of the neutrophil will be considered, together with discussion of recent advances in understanding neutrophil function and the factors that influence them throughout the phases of inflammation in ARDS. From a better understanding of neutrophils in this context, potential therapeutic targets are identified and discussed. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Neutrófilos/fisiologia , Pneumonia/fisiopatologia , Doença Aguda , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Citocinas/fisiologia , Modelos Animais de Doenças , Armadilhas Extracelulares/fisiologia , Humanos , Integrinas/fisiologia , Metaloproteinases da Matriz/fisiologia , Camundongos , Tecido Parenquimatoso/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Síndrome do Desconforto Respiratório/fisiopatologia , Selectinas/fisiologiaRESUMO
BACKGROUND: Eosinophils play a central role in propagation of allergic diseases, including asthma. Both recruitment and retention of eosinophils regulate pulmonary eosinophilia, but the question of whether alterations in apoptotic cell clearance by phagocytes contributes directly to resolution of allergic airway inflammation remains unexplored. OBJECTIVES: In this study we investigated the role of the receptor tyrosine kinase Mer in mediating apoptotic eosinophil clearance and allergic airway inflammation resolution in vivo to establish whether apoptotic cell clearance directly affects the resolution of allergic airway inflammation. METHODS: Alveolar and bone marrow macrophages were used to study Mer-mediated phagocytosis of apoptotic eosinophils. Allergic airway inflammation resolution was modeled in mice by using ovalbumin. Fluorescently labeled apoptotic cells were administered intratracheally or eosinophil apoptosis was driven by administration of dexamethasone to determine apoptotic cell clearance in vivo. RESULTS: Inhibition or absence of Mer impaired phagocytosis of apoptotic human and mouse eosinophils by macrophages. Mer-deficient mice showed delayed resolution of ovalbumin-induced allergic airway inflammation, together with increased airway responsiveness to aerosolized methacholine, increased bronchoalveolar lavage fluid protein levels, altered cytokine production, and an excess of uncleared dying eosinophils after dexamethasone treatment. Alveolar macrophage phagocytosis was significantly Mer dependent, with the absence of Mer attenuating apoptotic cell clearance in vivo to enhance inflammation in response to apoptotic cells. CONCLUSIONS: We demonstrate that Mer-mediated apoptotic cell clearance by phagocytes contributes to resolution of allergic airway inflammation, suggesting that augmenting apoptotic cell clearance is a potential therapeutic strategy for treating allergic airway inflammation.
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Apoptose/imunologia , Eosinófilos/imunologia , Macrófagos/imunologia , Hipersensibilidade Respiratória/imunologia , c-Mer Tirosina Quinase/imunologia , Alérgenos/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/imunologia , Feminino , Humanos , Inflamação/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Fagocitose , c-Mer Tirosina Quinase/genéticaRESUMO
BACKGROUND: Cystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation. METHODS: Blood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR. RESULTS: CF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF. CONCLUSIONS: CF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.
Assuntos
Apoptose/fisiologia , Fibrose Cística/patologia , Armadilhas Extracelulares , Neutrófilos/fisiologia , Adulto , Animais , Estudos de Casos e Controles , Sobrevivência Celular , Fibrose Cística/sangue , Fibrose Cística/imunologia , Humanos , Inflamação , Suínos , Fatores de TempoRESUMO
OBJECTIVES: Mild traumatic brain injury in the form of concussion is extremely common, and the potential effects on pulmonary priming have been underestimated. The aim of this study was to characterize the pulmonary response following mild traumatic brain injury and assess the pulmonary susceptibility to lung injury after a subsequent innocuous pulmonary insult. DESIGN: Experimental in vivo study. SETTING: University research laboratory. SUBJECTS: Male CD1 mice. INTERVENTIONS: We developed a model of concussive traumatic brain injury in mice followed by pulmonary acid microaspiration. To assess the dependent role of neutrophils in mediating pulmonary injury, we specifically depleted neutrophils. MEASUREMENTS AND MAIN RESULTS: Lateral fluid percussion to the brain resulted in neuronal damage and neutrophil infiltration as well as extensive pulmonary interstitial neutrophil accumulation but no alveolar injury. Following subsequent innocuous acid microaspiration, augmented alveolar neutrophil influx led to the development of pulmonary hemorrhage that was reduced following neutrophil depletion. CONCLUSIONS: This model shows for the first time that innocuous acid microaspiration is sufficient to induce neutrophil-mediated lung injury following mild concussion and that the extracranial effects of mild traumatic brain injury have been underestimated.
Assuntos
Concussão Encefálica/complicações , Lesão Pulmonar/etiologia , Infiltração de Neutrófilos , Animais , Pulmão/imunologia , Pulmão/patologia , Masculino , CamundongosRESUMO
Acute respiratory distress syndrome (ARDS) is a neutrophil-dominant disorder with no effective pharmacological therapies. While the cyclin-dependent kinase inhibitor AT7519 induces neutrophil apoptosis to promote inflammation resolution in preclinical models of lung inflammation, its potential efficacy in ARDS has not been examined. Untreated peripheral blood sepsis-related ARDS neutrophils demonstrated prolonged survival after 20â hours in vitro culture. AT7519 was able to override this phenotype to induce apoptosis in ARDS neutrophils with reduced expression of the pro-survival protein Mcl-1. We demonstrate the first pharmacological compound to induce neutrophil apoptosis in sepsis-related ARDS, highlighting cyclin-dependent kinase inhibitors as potential novel therapeutic agents.
Assuntos
Apoptose/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Piperidinas/uso terapêutico , Pirazóis/uso terapêutico , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/etiologia , Sepse/complicações , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de SobrevidaRESUMO
BACKGROUND: Acute respiratory distress syndrome (ARDS) is an often fatal neutrophil-dominant lung disease. Although influenced by multiple proinflammatory mediators, identification of suitable therapeutic candidates remains elusive. We aimed to delineate the presence of mitochondrial formylated peptides in ARDS and characterise the functional importance of formyl peptide receptor 1 (FPR1) signalling in sterile lung inflammation. METHODS: Mitochondrial formylated peptides were identified in bronchoalveolar lavage fluid (BALF) and serum of patients with ARDS by liquid chromatography-tandem mass spectrometry. In vitro, human neutrophils were stimulated with mitochondrial formylated peptides and their effects assessed by flow cytometry and chemotaxis assay. Mouse lung injury was induced by mitochondrial formylated peptides or hydrochloric acid. Bone marrow chimeras determined the contribution of myeloid and parenchymal FPR1 to sterile lung inflammation. RESULTS: Mitochondrial formylated peptides were elevated in BALF and serum from patients with ARDS. These peptides drove neutrophil activation and chemotaxis through FPR1-dependent mechanisms in vitro and in vivo. In mouse lung injury, inflammation was attenuated in Fpr1-/- mice, effects recapitulated by a pharmacological FPR1 antagonist even when administered after the onset of injury. FPR1 expression was present in alveolar epithelium and chimeric mice demonstrated that both myeloid and parenchymal FPR1 contributed to lung inflammation. CONCLUSIONS: We provide the first definitive evidence of mitochondrial formylated peptides in human disease and demonstrate them to be elevated in ARDS and important in a mouse model of lung injury. This work reveals mitochondrial formylated peptide FPR1 signalling as a key driver of sterile acute lung injury and a potential therapeutic target in ARDS.
Assuntos
Receptores de Formil Peptídeo/imunologia , Síndrome do Desconforto Respiratório/imunologia , Animais , Líquido da Lavagem Broncoalveolar/química , Quimiotaxia de Leucócito/imunologia , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Camundongos , Mitocôndrias/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Espectrometria de Massas em TandemAssuntos
COVID-19/complicações , COVID-19/diagnóstico por imagem , Embolia Pulmonar/virologia , Trombose/diagnóstico por imagem , Trombose/virologia , Angiografia por Tomografia Computadorizada , Feminino , Fluordesoxiglucose F18 , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Embolia Pulmonar/diagnóstico por imagem , Compostos RadiofarmacêuticosRESUMO
Neutrophil migration to sites of inflammation and the subsequent execution of multiple functions are designed to contain and kill invading pathogens. These highly regulated and orchestrated processes are controlled by interactions between numerous receptors and their cognate ligands. Unraveling and identifying those that are central to inflammatory processes may represent novel therapeutic targets for the treatment of neutrophil-dominant inflammatory disorders in which dysregulated neutrophil recruitment, function, and elimination serve to potentiate rather than resolve an initial inflammatory insult. The first G protein-coupled receptor to be described on human neutrophils, formyl peptide receptor 1 (FPR1), is one such receptor that plays a significant role in the execution of these functions through multiple intracellular signaling pathways. Recent work has highlighted important observations with regard to both receptor function and the importance and functional relevance of FPR1 in the pathogenesis of a range of both sterile and infective inflammatory conditions. In this review, we explore the multiple components of neutrophil migration and function in both health and disease, with a focus on the role of FPR1 in these processes. The current understanding of FPR1 structure, function, and signaling is examined, alongside discussion of the potential importance of FPR1 in inflammatory diseases suggesting that FPR1 is a key regulator of the inflammatory environment.
Assuntos
Inflamação/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Receptores de Formil Peptídeo/imunologia , Animais , Quimiotaxia de Leucócito/imunologia , Humanos , Ativação de Neutrófilo/imunologiaRESUMO
RATIONALE: Eosinophils are key effector cells in allergic diseases, including allergic rhinitis, eczema, and asthma. Their tissue presence is regulated by both recruitment and increased longevity at inflamed sites. OBJECTIVES: To investigate the ability of the flavone wogonin to induce eosinophil apoptosis in vitro and attenuate eosinophil-dominant allergic inflammation in vivo in mice. METHODS: Human and mouse eosinophil apoptosis in response to wogonin was investigated by cellular morphology, flow cytometry, mitochondrial membrane permeability, and pharmacological caspase inhibition. Allergic lung inflammation was modeled in mice sensitized and challenged with ovalbumin. Bronchoalveolar lavage (BAL) and lung tissue were examined for inflammation, mucus production, and inflammatory mediator production. Airway hyperresponsiveness to aerosolized methacholine was measured. MEASUREMENTS AND MAIN RESULTS: Wogonin induced time- and concentration-dependent human and mouse eosinophil apoptosis in vitro. Wogonin-induced eosinophil apoptosis occurred with activation of caspase-3 and was inhibited by pharmacological caspase inhibition. Wogonin administration attenuated allergic airway inflammation in vivo with reductions in BAL and interstitial eosinophil numbers, increased eosinophil apoptosis, reduced airway mucus production, and attenuated airway hyperresponsiveness. This wogonin-induced reduction in allergic airway inflammation was prevented by concurrent caspase inhibition in vivo. CONCLUSIONS: Wogonin induces eosinophil apoptosis and attenuates allergic airway inflammation, suggesting that it has therapeutic potential for the treatment of allergic inflammation in humans.
Assuntos
Apoptose/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Flavanonas/farmacologia , Hipersensibilidade/fisiopatologia , Animais , Lavagem Broncoalveolar , Eosinófilos/citologia , Feminino , Citometria de Fluxo , Humanos , Técnicas In Vitro , Inflamação , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologiaAssuntos
Neoplasias Pulmonares/diagnóstico , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologiaRESUMO
Neutrophil apoptosis and subsequent nonphlogistic clearance by surrounding phagocytes are key to the successful resolution of neutrophilic inflammation, with dysregulated apoptosis reported in multiple human inflammatory diseases. Enhancing neutrophil apoptosis has proresolution and anti-inflammatory effects in preclinical models of inflammation. Here we investigate the ability of the flavones apigenin, luteolin, and wogonin to induce neutrophil apoptosis in vitro and resolve neutrophilic inflammation in vivo. Human neutrophil apoptosis was assessed morphologically and by flow cytometry following incubation with apigenin, luteolin, and wogonin. All three flavones induced time- and concentration-dependent neutrophil apoptosis (apigenin, EC=12.2 µM; luteolin, EC=14.6 µM; and wogonin, EC=28.9 µM). Induction of apoptosis was caspase dependent, as it was blocked by the broad-spectrum caspase inhibitor Q-VD-OPh and was associated with both caspase-3 and caspase-9 activation. Flavone-induced apoptosis was preceded by down-regulation of the prosurvival protein Mcl-1, with proteasomal inhibition preventing flavone-induced Mcl-1 down-regulation and apoptosis. The flavones abrogated the survival effects of mediators that prolong neutrophil life span, including lipoteichoic acid, peptidoglycan, dexamethasone, and granulocyte-macrophage colony stimulating factor, by driving apoptosis. Furthermore, wogonin enhanced resolution of established neutrophilic inflammation in a zebrafish model of sterile tissue injury. Wogonin-induced resolution was dependent on apoptosis in vivo as it was blocked by caspase inhibition. Our data show that the flavones induce neutrophil apoptosis and have potential as neutrophil apoptosis-inducing anti-inflammatory, proresolution agents.
Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Flavonas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Inibidores de Caspase/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Proteína de Sequência 1 de Leucemia de Células Mieloides , Quinolinas/farmacologia , Peixe-ZebraRESUMO
Label-free autofluorescence lifetime is a unique feature of the inherent fluorescence signals emitted by natural fluorophores in biological samples. Fluorescence lifetime imaging microscopy (FLIM) can capture these signals enabling comprehensive analyses of biological samples. Despite the fundamental importance and wide application of FLIM in biomedical and clinical sciences, existing methods for analysing FLIM images often struggle to provide rapid and precise interpretations without reliable references, such as histology images, which are usually unavailable alongside FLIM images. To address this issue, we propose a deep learning (DL)-based approach for generating virtual Hematoxylin and Eosin (H&E) staining. By combining an advanced DL model with a contemporary image quality metric, we can generate clinical-grade virtual H&E-stained images from label-free FLIM images acquired on unstained tissue samples. Our experiments also show that the inclusion of lifetime information, an extra dimension beyond intensity, results in more accurate reconstructions of virtual staining when compared to using intensity-only images. This advancement allows for the instant and accurate interpretation of FLIM images at the cellular level without the complexities associated with co-registering FLIM and histology images. Consequently, we are able to identify distinct lifetime signatures of seven different cell types commonly found in the tumour microenvironment, opening up new opportunities towards biomarker-free tissue histology using FLIM across multiple cancer types.
RESUMO
Lung cancer is the most common cause of cancer-related deaths worldwide. Early detection improves outcomes, however, existing sampling techniques are associated with suboptimal diagnostic yield and procedure-related complications. Autofluorescence-based fluorescence-lifetime imaging microscopy (FLIM), a technique which measures endogenous fluorophore decay rates, may aid identification of optimal biopsy sites in suspected lung cancer. Our fibre-based fluorescence-lifetime imaging system, utilising 488 nm excitation, which is deliverable via existing diagnostic platforms, enables real-time visualisation and lifetime analysis of distal alveolar lung structure. We evaluated the diagnostic accuracy of the fibre-based fluorescence-lifetime imaging system to detect changes in fluorescence lifetime in freshly resected ex vivo lung cancer and adjacent healthy tissue as a first step towards future translation. The study compares paired non-small cell lung cancer (NSCLC) and non-cancerous tissues with gold standard diagnostic pathology to assess the performance of the technique. Paired NSCLC and non-cancerous lung tissues were obtained from thoracic resection patients (N=21). A clinically compatible 488 nm fluorescence-lifetime endomicroscopy platform was used to acquire simultaneous fluorescence intensity and lifetime images. Fluorescence lifetimes were calculated using a computationally-lightweight, rapid lifetime determination method. Fluorescence lifetime was significantly reduced in ex vivo lung cancer, compared with non-cancerous lung tissue [mean ± standard deviation (SD), 1.79±0.40 vs. 2.15±0.26 ns, P<0.0001], and fluorescence intensity images demonstrated distortion of alveolar elastin autofluorescence structure. Fibre-based fluorescence-lifetime imaging demonstrated good performance characteristics for distinguishing lung cancer, from adjacent non-cancerous tissue, with 81.0% sensitivity and 71.4% specificity. Our novel fibre-based fluorescence-lifetime imaging system, which enables label-free imaging and quantitative lifetime analysis, discriminates ex vivo lung cancer from adjacent healthy tissue. This minimally invasive technique has potential to be translated as a real-time biopsy guidance tool, capable of optimising diagnostic accuracy in lung cancer.
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Background: Formylated peptide receptor (FPR)-1 is a G-coupled receptor that senses foreign bacterial and host-derived mitochondrial formylated peptides (FPs), leading to innate immune system activation. Aim: We sought to investigate the role of FPR1-mediated inflammation and its potential as a therapeutic target in inflammatory bowel disease (IBD). Methods: We characterized FPR1 gene and protein expression in 8 human IBD (~1000 patients) datasets with analysis on disease subtype, mucosal inflammation, and drug response. We performed in vivo dextran-sulfate sodium (DSS) colitis in C57/BL6 FPR1 knockout mice. In ex vivo studies, we studied the role of mitochondrial FPs and pharmacological blockade of FPR1 using cyclosporin H in human peripheral blood neutrophils. Finally, we assess mitochondrial FPs as a potential mechanistic biomarker in the blood and stools of patients with IBD. Results: Detailed in silico analysis in human intestinal biopsies showed that FPR1 is highly expressed in IBD (nâ =â 207 IBD vs 67 non-IBD controls, Pâ <â .001), and highly correlated with gut inflammation in ulcerative colitis (UC) and Crohn's disease (CD) (both Pâ <â .001). FPR1 receptor is predominantly expressed in leukocytes, and we showed significantly higher FPR1+ve neutrophils in inflamed gut tissue section in IBD (17 CD and 24 UC; both Pâ <â .001). Further analysis in 6 independent IBD (data available under Gene Expression Omnibus accession numbers GSE59071, GSE206285, GSE73661, GSE16879, GSE92415, and GSE235970) showed an association with active gut inflammation and treatment resistance to infliximab, ustekinumab, and vedolizumab. FPR1 gene deletion is protective in murine DSS colitis with lower gut neutrophil inflammation. In the human ex vivo neutrophil system, mitochondrial FP, nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) is a potent activator of neutrophils resulting in higher CD62L shedding, CD63 expression, reactive oxygen species production, and chemotactic capacity; these effects are inhibited by cyclosporin H. We screened for mitochondrial ND6 in IBD (nâ =â 54) using ELISA and detected ND6 in stools with median values of 2.2 gg/mL (interquartile range [IQR] 0.0-4.99; range 0-53.3) but not in blood. Stool ND6 levels, however, were not significantly correlated with paired stool calprotectin, C-reactive protein, and clinical IBD activity. Conclusions: Our data suggest that FPR1-mediated neutrophilic inflammation is a tractable target in IBD; however, further work is required to clarify the clinical utility of mitochondrial FPs as a potential mechanistic marker for future stratification.
RESUMO
Introduction. The identification of mitotic figures is essential for the diagnosis, grading, and classification of various different tumors. Despite its importance, there is a paucity of literature reporting the consistency in interpreting mitotic figures among pathologists. This study leverages publicly accessible datasets and social media to recruit an international group of pathologists to score an image database of more than 1000 mitotic figures collectively. Materials and Methods. Pathologists were instructed to randomly select a digital slide from The Cancer Genome Atlas (TCGA) datasets and annotate 10-20 mitotic figures within a 2â mm2 area. The first 1010 submitted mitotic figures were used to create an image dataset, with each figure transformed into an individual tile at 40x magnification. The dataset was redistributed to all pathologists to review and determine whether each tile constituted a mitotic figure. Results. Overall pathologists had a median agreement rate of 80.2% (range 42.0%-95.7%). Individual mitotic figure tiles had a median agreement rate of 87.1% and a fair inter-rater agreement across all tiles (kappa = 0.284). Mitotic figures in prometaphase had lower percentage agreement rates compared to other phases of mitosis. Conclusion. This dataset stands as the largest international consensus study for mitotic figures to date and can be utilized as a training set for future studies. The agreement range reflects a spectrum of criteria that pathologists use to decide what constitutes a mitotic figure, which may have potential implications in tumor diagnostics and clinical management.