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Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1-mediated epithelial-mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-ß-induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial-mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1-tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-ß-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor-like repeats. Together, these data identify that aberrant bidirectional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.
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Transição Epitelial-Mesenquimal/fisiologia , Fibrose Pulmonar Idiopática/fisiopatologia , Movimento Celular , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibrose/fisiopatologia , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/patologia , Masculino , Cultura Primária de Células , Fibrose Pulmonar/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoAssuntos
Infecções por Coronavirus/terapia , Ventilação não Invasiva/métodos , Pneumonia Viral/terapia , Insuficiência Respiratória/terapia , Doença Aguda , Adolescente , Adulto , Idoso , COVID-19 , Estudos de Coortes , Infecções por Coronavirus/complicações , Cuidados Críticos/métodos , Feminino , Humanos , Hipóxia/etiologia , Hipóxia/terapia , Masculino , Pessoa de Meia-Idade , Ventilação não Invasiva/efeitos adversos , Pandemias , Pneumonia Viral/complicações , Respiração Artificial/efeitos adversos , Respiração Artificial/métodos , Insuficiência Respiratória/etiologia , Adulto JovemRESUMO
BACKGROUND: The success of early dexamethasone therapy for hospitalised COVID-19 cases in treatment of Sars-CoV-2 infection may predominantly reflect its anti-inflammatory action against a hyperinflammation (HI) response. It is likely that there is substantial heterogeneity in HI responses in COVID-19. METHODS: Blood CRP, ferritin, neutrophil, lymphocyte and platelet counts were scored to assess HI (HI5) and combined with a validated measure of generalised medical deterioration (NEWS2) before day 2. Our primary outcome was 28 day mortality from early treatment with dexamethasone stratified by HI5-NEWS2 status. FINDINGS: Of 1265 patients, high risk of HI (high HI5-NEWS2) (n = 367, 29.0%) conferred a strikingly increased mortality (36.0% vs 7.8%; Age adjusted hazard ratio (aHR) 5.9; 95% CI 3.6-9.8, p<0.001) compared to the low risk group (n = 455, 36.0%). An intermediate risk group (n = 443, 35.0%) also showed significantly higher mortality than the low risk group (17.6% vs 7.8%), aHR 2.2, p = 0.005). Early dexamethasone treatment conferred a 50.0% reduction in mortality in the high risk group (36.0% to 18.0%, aHR 0.56, p = 0.007). The intermediate risk group showed a trend to reduction in mortality (17.8% to 10.3%, aHR 0.82, p = 0.46) which was not observed in the low risk group (7.8% to 9.2%, aHR 1.4, p = 0.31). INTERPRETATION: Higher HI5-NEWS2 scores measured at COVID-19 diagnosis, strongly associate with increased mortality at 28 days. Significant reduction in mortality with early dexamethasone treatment was only observed in the high risk group. Therefore, the HI5-NEWS2 score could be utilised to stratify randomised clinical trials to test whether intensified anti-inflammatory therapy would further benefit high risk patients and whether alternative approaches would benefit low risk groups. Considering its recognised morbidity, we suggest that early dexamethasone should not be routinely prescribed for HI5-NEWS2 low risk individuals with COVID-19 and clinicians should cautiously assess the risk benefit of this intervention in all cases.
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COVID-19 , Humanos , SARS-CoV-2 , Teste para COVID-19 , Tratamento Farmacológico da COVID-19 , Anti-Inflamatórios/uso terapêutico , Dexametasona/uso terapêuticoRESUMO
BACKGROUND: Rituximab is often used as rescue therapy in interstitial lung disease (ILD) associated with connective tissue disease (CTD), but has not been studied in clinical trials. This study aimed to assess whether rituximab is superior to cyclophosphamide as a treatment for severe or progressive CTD associated ILD. METHODS: We conducted a randomised, double-blind, double-dummy, phase 2b trial to assess the superiority of rituximab compared with cyclophosphamide. Patients aged 18-80 years with severe or progressive ILD related to scleroderma, idiopathic inflammatory myositis, or mixed CTD, recruited across 11 specialist ILD or rheumatology centres in the UK, were randomly assigned (1:1) to receive rituximab (1000 mg at weeks 0 and 2 intravenously) or cyclophosphamide (600 mg/m2 body surface area every 4 weeks intravenously for six doses). The primary endpoint was rate of change in forced vital capacity (FVC) at 24 weeks compared with baseline, analysed using a mixed-effects model with random intercepts, adjusted for baseline FVC and CTD type. Prespecified secondary endpoints reported in this Article were change in FVC at 48 weeks versus baseline; changes from baseline in 6 min walk distance, diffusing capacity of the lung for carbon monoxide (DLCO), physician-assessed global disease activity (GDA) score, and quality-of-life scores on the St George's Respiratory Questionnaire (SGRQ), King's Brief Interstitial Lung Disease (KBILD) questionnaire, and European Quality of Life Five-Dimension (EQ-5D) questionnaire at 24 and 48 weeks; overall survival, progression-free survival, and time to treatment failure; and corticosteroid use. All endpoints were analysed in the modified intention-to-treat population, which comprised all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov (NCT01862926). FINDINGS: Between Dec 1, 2014, and March 31, 2020, we screened 145 participants, of whom 101 participants were randomly allocated: 50 (50%) to receive cyclophosphamide and 51 (50%) to receive rituximab. 48 (96%) participants in the cyclophosphamide group and 49 (96%) in the rituximab group received at least one dose of treatment and were included in analyses; 43 (86%) participants in the cyclophosphamide group and 42 (82%) participants in the rituximab group completed 24 weeks of treatment and follow-up. At 24 weeks, FVC was improved from baseline in both the cyclophosphamide group (unadjusted mean increase 99 mL [SD 329]) and the rituximab group (97 mL [234]); in the adjusted mixed-effects model, the difference in the primary endpoint at 24 weeks was -40 mL (95% CI -153 to 74; p=0·49) between the rituximab group and the cyclophosphamide group. KBILD quality-of-life scores were improved at 24 weeks by a mean 9·4 points (SD 20·8) in the cyclophosphamide group and 8·8 points (17·0) in the rituximab group. No significant differences in secondary endpoints were identified between the treatment groups, with the exception of change in GDA score at week 48, which favoured cyclophosphamide (difference 0·90 [95% CI 0·11 to 1·68]). Improvements in lung function and respiratory-related quality-of-life measures were observed in both treatment groups. Lower corticosteroid exposure over 48 weeks of follow-up was recorded in the rituximab group. Two (4%) of 48 participants who received cyclophosphamide and three (6%) of 49 who received rituximab died during the study, all due to complications of CTD or ILD. Overall survival, progression-free survival, and time to treatment failure did not significantly differ between the two groups. All participants reported at least one adverse event during the study. Numerically fewer adverse events were reported by participants receiving rituximab (445 events) than those receiving cyclophosphamide (646 events). Gastrointestinal and respiratory disorders were the most commonly reported adverse events in both groups. There were 62 serious adverse events of which 33 occurred in the cyclophosphamide group and 29 in the rituximab group. INTERPRETATION: Rituximab was not superior to cyclophosphamide to treat patients with CTD-ILD, although participants in both treatment groups had increased FVC at 24 weeks, in addition to clinically important improvements in patient-reported quality of life. Rituximab was associated with fewer adverse events. Rituximab should be considered as a therapeutic alternative to cyclophosphamide in individuals with CTD-ILD requiring intravenous therapy. FUNDING: Efficacy and Mechanism Evaluation Programme (Medical Research Council and National Institute for Health Research, UK).
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Doenças do Tecido Conjuntivo , Doenças Pulmonares Intersticiais , Humanos , Rituximab/uso terapêutico , Rituximab/efeitos adversos , Qualidade de Vida , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/etiologia , Ciclofosfamida/efeitos adversos , Doenças do Tecido Conjuntivo/complicações , Doenças do Tecido Conjuntivo/tratamento farmacológico , Doenças do Tecido Conjuntivo/induzido quimicamente , Corticosteroides/uso terapêutico , Método Duplo-Cego , Reino Unido , Resultado do TratamentoRESUMO
Exercise training is recommended for patients with idiopathic pulmonary fibrosis (IPF); however, the mechanism(s) underlying its physiological benefits remain unclear. We investigated the effects of an individualised aerobic interval training programme on exercise capacity and redox status in IPF patients. IPF patients were recruited prospectively to an 8-week, twice-weekly cardiopulmonary exercise test (CPET)-derived structured responsive exercise training programme (SRETP). Systemic redox status was assessed pre- and post-CPET at baseline and following SRETP completion. An age- and sex-matched non-IPF control cohort was recruited for baseline comparison only. At baseline, IPF patients (n = 15) had evidence of increased oxidative stress compared with the controls as judged by; the plasma reduced/oxidised glutathione ratio (median, control 1856 vs. IPF 736 p = 0.046). Eleven IPF patients completed the SRETP (median adherence 88%). Following SRETP completion, there was a significant improvement in exercise capacity assessed via the constant work-rate endurance time (+82%, p = 0.003). This was accompanied by an improvement in post-exercise redox status (in favour of antioxidants) assessed via serum total free thiols (median increase, +0.26 µmol/g protein p = 0.005) and total glutathione concentration (+0.73 µM p = 0.03), as well as a decrease in post-exercise lipid peroxidation products (-1.20 µM p = 0.02). Following SRETP completion, post-exercise circulating nitrite concentrations were significantly lower compared with baseline (-0.39 µM p = 0.04), suggestive of exercise-induced nitrite utilisation. The SRETP increased both endurance time and systemic antioxidant capacity in IPF patients. The observed reduction in nitrite concentrations provides a mechanistic rationale to investigate nitrite/nitrate supplementation in IPF patients.
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A defining pathological feature of human lung fibrosis is localized tissue heterogeneity, which challenges the interpretation of transcriptomic studies that typically lose spatial information. Here we investigate spatial gene expression in diagnostic tissue using digital profiling technology. We identify distinct, region-specific gene expression signatures as well as shared gene signatures. By integration with single-cell data, we spatially map the cellular composition within and distant from the fibrotic niche, demonstrating discrete changes in homeostatic and pathologic cell populations even in morphologically preserved lung, while through ligand-receptor analysis, we investigate cellular cross-talk within the fibrotic niche. We confirm findings through bioinformatic, tissue, and in vitro analyses, identifying that loss of NFKB inhibitor zeta in alveolar epithelial cells dysregulates the TGFß/IL-6 signaling axis, which may impair homeostatic responses to environmental stress. Thus, spatially resolved deconvolution advances understanding of cell composition and microenvironment in human lung fibrogenesis.
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Fibrose Pulmonar , Células Epiteliais Alveolares/metabolismo , Fibrose , Humanos , Pulmão/patologia , Fibrose Pulmonar/metabolismo , Transdução de SinaisRESUMO
Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFß increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.
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Colágeno/fisiologia , Fibrose Pulmonar/metabolismo , Biomarcadores , Células Cultivadas , Colágeno/química , Fibroblastos/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Fator 1 Induzível por Hipóxia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic scarring disease in which aging, environmental exposure(s) and genetic susceptibility have been implicated in disease pathogenesis, however, the causes and mechanisms of the progressive fibrotic cascade are still poorly understood. As epithelial-mesenchymal interactions are essential for normal wound healing, through human 2D and 3D in vitro studies, we tested the hypothesis that IPF fibroblasts (IPFFs) dysregulate alveolar epithelial homeostasis. Conditioned media from IPFFs exaggerated the wound-healing response of primary human Type II alveolar epithelial cells (AECs). Furthermore, AECs co-cultured with IPFFs exhibited irregular epithelialization compared with those co-cultured with control fibroblasts (NHLFs) or AECs alone, suggesting that epithelial homeostasis is dysregulated in IPF as a consequence of the abnormal secretory phenotype of IPFFs. Secretome analysis of IPFF conditioned media and functional studies identified the matricellular protein, SPARC, as a key mediator in the epithelial-mesenchymal paracrine signaling, with increased secretion of SPARC by IPFFs promoting persistent activation of alveolar epithelium via an integrin/focal adhesion/cellular-junction axis resulting in disruption of epithelial barrier integrity and increased macromolecular permeability. These findings suggest that in IPF fibroblast paracrine signaling promotes persistent alveolar epithelial activation, so preventing normal epithelial repair responses and restoration of tissue homeostasis. Furthermore, they identify SPARC-mediated paracrine signaling as a potential therapeutic target to promote the restoration of lung epithelial homoestasis in IPF patients.
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For patients with IPF, length of time in healthcare systems prior to review in an ILD clinic reflects disease severity and may impact upon patient outcome https://bit.ly/2TkO26r.
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The contribution of epithelial-mesenchymal transition (EMT) to human lung fibrogenesis is controversial. Here we provide evidence that ZEB1-mediated EMT in human alveolar epithelial type II (ATII) cells contributes to the development of lung fibrosis by paracrine signalling to underlying fibroblasts. Activation of EGFR-RAS-ERK signalling in ATII cells induced EMT via ZEB1. ATII cells had extremely low extracellular matrix gene expression even after induction of EMT, however conditioned media from ATII cells undergoing RAS-induced EMT augmented TGFß-induced profibrogenic responses in lung fibroblasts. This epithelial-mesenchymal crosstalk was controlled by ZEB1 via the expression of tissue plasminogen activator (tPA). In human fibrotic lung tissue, nuclear ZEB1 expression was detected in alveolar epithelium adjacent to sites of extracellular matrix (ECM) deposition, suggesting that ZEB1-mediated paracrine signalling has the potential to contribute to early fibrotic changes in the lung interstitium. Targeting this novel ZEB1 regulatory axis may be a viable strategy for the treatment of pulmonary fibrosis.
Assuntos
Diferenciação Celular/genética , Fibrose/genética , Doenças Respiratórias/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Fibrose/patologia , Regulação da Expressão Gênica/genética , Humanos , Pulmão/metabolismo , Pulmão/patologia , Miofibroblastos/metabolismo , Comunicação Parácrina/genética , Doenças Respiratórias/patologiaRESUMO
Idiopathic pulmonary fibrosis (IPF), the prototypic progressive fibrotic interstitial lung disease, is thought to be a consequence of repetitive micro-injuries to an ageing, susceptible alveolar epithelium. Ageing is a risk factor for IPF and incidence has been demonstrated to increase with age. Decreased (macro)autophagy with age has been reported extensively in a variety of systems and diseases, including IPF. However, it is undetermined whether the role of faulty autophagy is causal or coincidental in the context of IPF. Here, we report that in alveolar epithelial cells inhibition of autophagy promotes epithelial-mesenchymal transition (EMT), a process implicated in embryonic development, wound healing, cancer metastasis and fibrosis. We further demonstrate that this is attained, at least in part, by increased p62/SQSTM1 expression that promotes p65/RELA mediated-transactivation of an EMT transcription factor, Snail2 (SNAI2), which not only controls EMT but also regulates the production of locally acting profibrogenic mediators. Our data suggest that reduced autophagy induces EMT of alveolar epithelial cells and can contribute to fibrosis via aberrant epithelial-fibroblast crosstalk.
Assuntos
Transição Epitelial-Mesenquimal/genética , Fibrose Pulmonar Idiopática/genética , Proteína Sequestossoma-1/genética , Fatores de Transcrição da Família Snail/genética , Fator de Transcrição RelA/genética , Células A549 , Envelhecimento/genética , Envelhecimento/patologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Autofagia/genética , Diferenciação Celular/genética , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Cultura Primária de Células , Fatores de Risco , Fatores de TranscriçãoRESUMO
In IPF, commencement of antifibrotic therapy in patients with preserved lung volume may increase the duration of therapy http://ow.ly/4HeM30lFS67.
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Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.
Assuntos
Aminoácido Oxirredutases/antagonistas & inibidores , Colágeno/química , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/química , Fibrose Pulmonar/tratamento farmacológico , Reticulina/química , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Aminoácidos/química , Animais , Fenômenos Biomecânicos , Estudos de Casos e Controles , Colágeno/metabolismo , Colágeno/ultraestrutura , Reagentes de Ligações Cruzadas/química , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica , Homeostase/genética , Humanos , Pulmão/metabolismo , Pulmão/patologia , Mecanotransdução Celular , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Proteína-Lisina 6-Oxidase , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Reticulina/metabolismo , Reticulina/ultraestrutura , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Occupational asbestos exposure has been endemic in Portsmouth. A retrospective case note analysis of 50 patients who underwent thoracoscopy over a 2-year period from January 2003 was undertaken. Biopsies were taken in 47 cases, 31 of which showed malignant mesothelioma. Thirty seven percent of those without a history of direct exposure to asbestos had mesothelioma, implying that even in the absence of an exposure history a low threshold for investigation should be adopted for the local population. There was no mortality or significant morbidity associated with the procedure. Medical thoracoscopy is safe and effective in the diagnosis of benign and malignant pleural disease particularly in this high risk population.