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Respiratory virus infections in humans cause a broad-spectrum of diseases that result in substantial morbidity and mortality annually worldwide. To reduce the global burden of respiratory viral diseases, preventative and therapeutic interventions that are accessible and effective are urgently needed, especially in countries that are disproportionately affected. Repurposing generic medicine has the potential to bring new treatments for infectious diseases to patients efficiently and equitably. In this study, we found that intranasal delivery of neomycin, a generic aminoglycoside antibiotic, induces the expression of interferon-stimulated genes (ISGs) in the nasal mucosa that is independent of the commensal microbiota. Prophylactic or therapeutic administration of neomycin provided significant protection against upper respiratory infection and lethal disease in a mouse model of COVID-19. Furthermore, neomycin treatment protected Mx1 congenic mice from upper and lower respiratory infections with a highly virulent strain of influenza A virus. In Syrian hamsters, neomycin treatment potently mitigated contact transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In healthy humans, intranasal application of neomycin-containing Neosporin ointment was well tolerated and effective at inducing ISG expression in the nose in a subset of participants. These findings suggest that neomycin has the potential to be harnessed as a host-directed antiviral strategy for the prevention and treatment of respiratory viral infections.
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Administração Intranasal , Antivirais , Neomicina , SARS-CoV-2 , Animais , Neomicina/farmacologia , Neomicina/administração & dosagem , Camundongos , Humanos , Antivirais/farmacologia , Antivirais/administração & dosagem , SARS-CoV-2/imunologia , SARS-CoV-2/efeitos dos fármacos , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/virologia , Infecções Respiratórias/prevenção & controle , Mucosa Nasal/imunologia , Mucosa Nasal/virologia , Mucosa Nasal/efeitos dos fármacos , Modelos Animais de Doenças , Tratamento Farmacológico da COVID-19 , Mesocricetus , Feminino , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/imunologiaRESUMO
RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease for which current treatment options only slow clinical progression. Previously, we identified a subset of patients with IPF with an accelerated disease course associated with fibroblast expression of Toll-Like Receptor 9 (TLR9) mediated by interactions with its ligand mitochondrial DNA (mtDNA). OBJECTIVES: We aimed to show that TLR9 activation induces fibroproliferative responses that are abrogated by its antagonism by using two commercially-available indirect inhibitors and a proprietary, selective direct small molecule inhibitor. METHODS: We employed two independent cohorts of patients with IPF, multiple in vitro fibroblast cell culture platforms, an in vivo mouse model, and an ex vivo human precision cut lung slices system to investigate the clinical and biologic significance of TLR9 in this disease. MEASUREMENTS AND MAIN RESULTS: In two independent IPF cohorts, plasma mtDNA activates TLR9 in a manner associated with the expression of MCP-1, IL-6, TNFα, and IP-10 and worsened transplant-free survival. Our cell culture platform showed that TLR9 mediates fibroblast activation via TGFß1 and stiff substrates, and that its antagonism, particularly direct inhibition, ameliorates this process, including production of these TLR9 associated pharmacodynamic endpoints. We further demonstrated that direct TLR9 inhibition mitigates these fibroproliferative responses in our in vivo and ex vivo models of pulmonary fibrosis. CONCLUSIONS: In this novel study, we found that direct TLR9 inhibition mitigates fibroproliferative responses in preclinical models of pulmonary fibrosis. Our work demonstrates the therapeutic potential of direct TLR9 antagonism in IPF and related fibrotic lung diseases.
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Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5' single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P = 0.007), although not different than controls, and may be associated with decreased survival (P = 0.009 in Kaplan-Meier analysis; and P = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).
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Fibrose Pulmonar Idiopática , Leucócitos Mononucleares , Análise de Célula Única , Linfócitos T Reguladores , Humanos , Fibrose Pulmonar Idiopática/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Análise de Célula Única/métodos , Linfócitos T Reguladores/imunologia , Leucócitos Mononucleares/imunologia , Progressão da Doença , Estudos de Casos e Controles , Citometria de FluxoRESUMO
Rationale: Fibrotic hypersensitivity pneumonitis is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives: To elucidate immune aberrations in fibrotic hypersensitivity pneumonitis in single-cell resolution. Methods: Single-cell 5' RNA sequencing was conducted on peripheral blood mononuclear cells and bronchoalveolar lavage cells obtained from 45 patients with fibrotic hypersensitivity pneumonitis, 63 idiopathic pulmonary fibrosis, 4 non-fibrotic hypersensitivity pneumonitis, and 36 healthy controls in the United States and Mexico. Analyses included differential gene expression (Seurat), transcription factor activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3/Velocyto-scVelo-CellRank). Measurements and Main Results: Overall, 501,534 peripheral blood mononuclear cells from 110 patients and controls and 88,336 bronchoalveolar lavage cells from 19 patients were profiled. Compared to controls, fibrotic hypersensitivity pneumonitis has elevated classical monocytes (adjusted-p=2.5e-3) and are enriched in CCL3hi/CCL4hi and S100Ahi classical monocytes (adjusted-p<2.2e-16). Trajectory analyses demonstrate that S100Ahi classical monocytes differentiate into SPP1hi lung macrophages associated with fibrosis. Compared to both controls and idiopathic pulmonary fibrosis, fibrotic hypersensitivity pneumonitis patient cells are significantly enriched in GZMhi cytotoxic T cells. These cells exhibit transcription factor activities indicative of TGFß and TNFα/NFκB pathways. These results are publicly available at https://ildimmunecellatlas.org. Conclusions: Single-cell transcriptomics of fibrotic hypersensitivity pneumonitis patients uncovered novel immune perturbations, including previously undescribed increases in GZMhi cytotoxic CD4+ and CD8+ T cells - reflecting this disease's unique inflammatory T-cell driven nature - as well as increased S100Ahi and CCL3hi/CCL4hi classical monocytes also observed in idiopathic pulmonary fibrosis. Both cell populations may guide the development of new biomarkers and therapeutic interventions.
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INTRODUCTION: The pathogenesis of sarcoidosis involves tissue remodelling mediated by the accumulation of abnormal extracellular matrix, which is partly the result of an imbalance in collagen synthesis, cross-linking and degradation. During this process, collagen fragments or neoepitopes, are released into the circulation. The significance of these circulating collagen neoepitopes in sarcoidosis remains unknown. METHODS: We employed plasma samples from patients with sarcoidosis enrolled in A Case Control Etiologic Study of Sarcoidosis (ACCESS) and Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS), and healthy control patients recruited from the Yale community. Plasma concentrations of type III and VI collagen degradation (C3M and C6M) and formation (PRO-C3 and PRO-C6) were quantified via neoepitope-specific competitive ELISA, and statistical associations were sought with clinical phenotypes. RESULTS: Relative to healthy controls, the plasma of both sarcoidosis cohorts was enriched for C3M and C6M, irrespective of corticosteroid use and disease duration. While circulating collagen neoepitopes were independent of Scadding stage, there was a significant association between multiorgan disease and PRO-C3, PRO-C6 and C3M in the ACCESS cohort; PRO-C3 and C6M displayed this property in GRADS. These findings were unrelated to plasma levels of interleukin-4 (IL-4), IL-5, IL-6, IL-9, IL-10 and IL-13. Moreover, PRO-C3 was associated with dermatological disease in both cohorts. DISCUSSION: In two well-characterised sarcoidosis cohorts, we discovered that the plasma is enriched for neoepitopes of collagen degradation (C3M and C6M). In multiorgan disease, there was an association with circulating neoepitopes of type III formation (PRO-C3), perhaps mediated by dermatological sarcoidosis. Further investigation in this arena has the potential to foster new insights into the pathogenic mechanisms of this complex disease.
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Idiopathic pulmonary fibrosis is increasingly associated with nerve-driven processes and endogenous innate immune ligands such as mitochondrial DNA (mtDNA). Interestingly, a connection between these entities has not been explored. Here, we report that noradrenaline (NA) derived from the lung's adrenergic nerve supply drives α-smooth muscle actin (αSMA)-expressing fibroblast accumulation via mechanisms involving α1 adrenoreceptors and mtDNA. Using the bleomycin model, we compared ablation of the lung's adrenergic nerve supply with surgical adrenal resection and found that NA derived from local but not adrenal sources contributes to experimentally induced lung fibrosis and the emergence of an αSMA+ve fibroblast population expressing adrenoreceptor α-1D (ADRA1D). Therapeutic delivery of the α1 adrenoreceptor antagonist terazosin reversed these changes and suppressed extracellular mtDNA accumulation. Cultured normal human lung fibroblasts displayed α1 adrenoreceptors and in response to costimulation with TGFß1 and NA adopted ACTA2 expression and extracellular mtDNA release. These findings were opposed by terazosin. Evaluation of a previously studied IPF cohort revealed that patients prescribed α1 adrenoreceptor antagonists for nonpulmonary indications demonstrated improved survival and reduced concentrations of plasma mtDNA. Our observations link nerve-derived NA, α1 adrenoreceptors, extracellular mtDNA, and lung fibrogenesis in mouse models, cultured cells, and humans with IPF. Further study of this neuroinnate connection may yield new avenues for investigation in the clinical and basic science realms.
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DNA Mitocondrial , Fibrose Pulmonar Idiopática , Camundongos , Animais , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Transdução de Sinais , Fibroblastos/metabolismo , Bleomicina/farmacologia , Adrenérgicos/metabolismo , Adrenérgicos/farmacologiaRESUMO
Sarcoidosis is a chronic granulomatous disease of unknown etiology that primarily affects the lungs. The development of stage IV or fibrotic lung disease accounts for a significant proportion of the morbidity and mortality attributable to sarcoidosis. Further investigation into the active mechanisms of disease pathogenesis and fibrogenesis might illuminate fundamental mediators of injury and repair while providing new opportunities for clinical intervention. However, progress in sarcoidosis research has been hampered by the heterogeneity of clinical phenotypes and the lack of a consensus modeling system. Recently, reverse translational research, wherein observations made at the patient level catalyze hypothesis-driven research at the laboratory bench, has generated new discoveries regarding the immunopathogenic mechanisms of pulmonary granuloma formation, fibrogenesis, and disease model development. The purpose of this review is to highlight the promise and possibility of these novel investigative efforts.
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Fibrose Pulmonar , Sarcoidose , Granuloma/patologia , Humanos , Pulmão/patologia , Fibrose Pulmonar/patologia , Sarcoidose/patologia , Pesquisa Translacional BiomédicaRESUMO
Sarcoidosis is a systemic granulomatous disease predominantly affecting the lungs. The mechanisms promoting disease pathogenesis and progression are unknown, although interleukin-15 (IL-15) has been associated with the immune-mediated inflammation of sarcoidosis. Because the identification of a mechanistically based, clinically relevant biomarker for sarcoidosis remains elusive, we hypothesized this role for IL-15. Pulmonary sarcoidosis granuloma formation was modeled using trehalose 6,6'-dimicolate (TDM), which was administered into wild-type and three lineages of mice: those overexpressing IL-15, deficient in IL-15, and deficient in IL-15 receptor α. The number of granulomas per lung was counted and normalized to the wild type. IL-15 concentrations were measured in the bronchoalveolar lavage (BAL) from healthy controls and subjects with sarcoidosis in our cohort, where associations between IL-15 levels and clinical manifestations were sought. Findings were validated in another independent sarcoidosis cohort. TDM administration resulted in similar granuloma numbers across all lineages of mice. IL-15 concentrations were elevated in the BAL of both human cohorts, irrespective of disease phenotypes. In exploratory analysis, an association with obesity was observed, and various other soluble mediators were identified in the BAL of both cohorts. Although IL-15 is enriched in the sarcoidosis lung, it was independent of disease pathogenesis or clinical manifestations in our mouse model and human cohorts of sarcoidosis. An association with obesity perhaps reflects the ongoing inflammatory processes of these comorbid conditions. Our findings showed that IL-15 is redundant for disease pathogenesis and clinical progression of sarcoidosis.
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Granuloma/metabolismo , Interleucina-15/metabolismo , Fenótipo , Sarcoidose Pulmonar/patologia , Sarcoidose/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Granuloma/patologia , Inflamação/patologia , Interleucina-15/genética , Pulmão/metabolismo , Pulmão/patologia , Sarcoidose/patologia , Sarcoidose Pulmonar/complicaçõesRESUMO
BACKGROUND: Vascular smooth muscle cells (SMCs) synthesize extracellular matrix (ECM) that contributes to tissue remodeling after revascularization interventions. The cytokine transforming growth factor ß (TGF-ß) is induced on tissue injury and regulates tissue remodeling and wound healing, but dysregulated signaling results in excess ECM deposition and fibrosis. The LIM (Lin11, Isl-1 & Mec-3) domain protein LIM domain only 7 (LMO7) is a TGF-ß1 target gene in hepatoma cells, but its role in vascular physiology and fibrosis is unknown. METHODS: We use carotid ligation and femoral artery denudation models in mice with global or inducible smooth muscle-specific deletion of LMO7, and knockout, knockdown, overexpression, and mutagenesis approaches in mouse and human SMC, and human arteriovenous fistula and cardiac allograft vasculopathy samples to assess the role of LMO7 in neointima and fibrosis. RESULTS: We demonstrate that LMO7 is induced postinjury and by TGF-ß in SMC in vitro. Global or SMC-specific LMO7 deletion enhanced neointimal formation, TGF-ß signaling, ECM deposition, and proliferation in vascular injury models. LMO7 loss of function in human and mouse SMC enhanced ECM protein expression at baseline and after TGF-ß treatment. TGF-ß neutralization or receptor antagonism prevented the exacerbated neointimal formation and ECM synthesis conferred by loss of LMO7. Notably, loss of LMO7 coordinately amplified TGF-ß signaling by inducing expression of Tgfb1 mRNA, TGF-ß protein, αv and ß3 integrins that promote activation of latent TGF-ß, and downstream effectors SMAD3 phosphorylation and connective tissue growth factor. Mechanistically, the LMO7 LIM domain interacts with activator protein 1 transcription factor subunits c-FOS and c-JUN and promotes their ubiquitination and degradation, disrupting activator protein 1-dependent TGF-ß autoinduction. Importantly, preliminary studies suggest that LMO7 is upregulated in human intimal hyperplastic arteriovenous fistula and cardiac allograft vasculopathy samples, and inversely correlates with SMAD3 phosphorylation in cardiac allograft vasculopathy. CONCLUSIONS: LMO7 is induced by TGF-ß and serves to limit vascular fibrotic responses through negative feedback regulation of the TGF-ß pathway. This mechanism has important implications for intimal hyperplasia, wound healing, and fibrotic diseases.
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Proteínas com Domínio LIM/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Remodelação Vascular , Lesões do Sistema Vascular/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Retroalimentação Fisiológica , Fibrose , Hiperplasia , Integrina alfaVbeta3/metabolismo , Proteínas com Domínio LIM/deficiência , Proteínas com Domínio LIM/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/genética , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologiaRESUMO
Idiopathic pulmonary fibrosis (IPF) is the most common and devastating of the interstitial lung diseases. Epithelial dysfunction is thought to play a prominent role in disease pathology, and we sought to characterize secreted signals that may contribute to disease pathology. Transcriptional profiling of senescent type II alveolar epithelial cells from mice with epithelial-specific telomere dysfunction identified the transforming growth factor-ß family member, growth and differentiation factor 15 (Gdf15), as the most significantly upregulated secreted protein. Gdf15 expression is induced in response to telomere dysfunction and bleomycin challenge in mice. Gdf15 mRNA is expressed by lung epithelial cells, and protein can be detected in peripheral blood and bronchoalveolar lavage following bleomycin challenge in mice. In patients with IPF, GDF15 mRNA expression in lung tissue is significantly increased and correlates with pulmonary function. Single-cell RNA sequencing of human lungs identifies epithelial cells as the primary source of GDF15, and circulating concentrations of GDF15 are markedly elevated and correlate with disease severity and survival in multiple independent cohorts. Our findings suggest that GDF15 is an epithelial-derived secreted protein that may be a useful biomarker of epithelial stress and identifies IPF patients with poor outcomes.
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Células Epiteliais Alveolares/metabolismo , Fator 15 de Diferenciação de Crescimento/genética , Fibrose Pulmonar Idiopática/genética , Transcriptoma , Idoso , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/patologia , Animais , Bleomicina/administração & dosagem , Líquido da Lavagem Broncoalveolar/química , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Fibrose Pulmonar Idiopática/diagnóstico , Fibrose Pulmonar Idiopática/mortalidade , Fibrose Pulmonar Idiopática/fisiopatologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Testes de Função Respiratória , Índice de Gravidade de Doença , Análise de Sobrevida , TelômeroRESUMO
Sarcoidosis is an unpredictable granulomatous disease in which African Americans disproportionately experience aggressive phenotypes. Mitochondrial DNA (mtDNA) released by cells in response to various stressors contributes to tissue remodelling and inflammation. While extracellular mtDNA has emerged as a biomarker in multiple diseases, its relevance to sarcoidosis remains unknown. We aimed to define an association between extracellular mtDNA and clinical features of sarcoidosis.Extracellular mtDNA concentrations were measured using quantitative PCR for the human MT-ATP6 gene in bronchoalveolar (BAL) and plasma samples from healthy controls and patients with sarcoidosis from The Yale Lung Repository; associations between MT-ATP6 concentrations and Scadding stage, extrapulmonary disease and demographics were sought. Results were validated in the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis cohort.Relative to controls, MT-ATP6 concentrations in sarcoidosis subjects were robustly elevated in the BAL fluid and plasma, particularly in the plasma of patients with extrapulmonary disease. Relative to Caucasians, African Americans displayed excessive MT-ATP6 concentrations in the BAL fluid and plasma, for which the latter compartment correlated with significantly higher odds of extrapulmonary disease.Enrichments in extracellular mtDNA in sarcoidosis are associated with extrapulmonary disease and African American descent. Further study into the mechanistic basis of these clinical findings may lead to novel pathophysiologic and therapeutic insights.
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DNA Mitocondrial/sangue , Sarcoidose Pulmonar/sangue , Adulto , Idoso , Biomarcadores/sangue , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar , Estudos de Casos e Controles , Feminino , Células HEK293 , Proteína HMGB1/metabolismo , Humanos , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , ATPases Mitocondriais Próton-Translocadoras/sangue , Fenótipo , Sarcoidose Pulmonar/fisiopatologia , Receptor Toll-Like 9/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Molecular biomarkers are needed to refine prognostication and phenotyping of pulmonary hypertension (PH) patients. S100A12 is an emerging biomarker of various inflammatory diseases. This study aims to determine the prognostic value of S100A12 in PH. METHODS: Exploratory microarray analysis performed on peripheral blood mononuclear cells (PBMC) collected from idiopathic pulmonary fibrosis (IPF) patients suggested an association between S100A12 and both PH and mortality. So the current study was designed to evaluate for an association between S100A12 in peripheral blood collected from two well-phenotyped PH cohorts in two other centres to derive and validate an association between S100A12 protein serum concentrations and mortality. RESULTS: The majority of the patients in the discovery and validation cohorts were either World Health Organization (WHO) group 1 (pulmonary arterial hypertension (PAH)) or 3 (lung disease-associated) PH. In the discovery PH cohort, S100A12 was significantly increased in patients with PH (n = 51) compared to controls (n = 22) (29.8 vs 15.7 ng/mL, P < 0.001) and negatively correlated with cardiac output (r = -0.58, P < 0.001) in PH patients. When S100A12 data were pooled from both cohorts, PAH and non-PAH PH patients had higher S100A12 compared to healthy external controls (32.6, 30.9, 15.7 ng/mL; P < 0.001). S100A12 was associated with an increased risk in overall mortality in PH patients in both the discovery (n = 51; P = 0.008) and validation (n = 40; P < 0.001) cohorts. CONCLUSION: S100A12 levels are increased in PH patients and are associated with increased mortality.
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Débito Cardíaco , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/mortalidade , Proteína S100A12/sangue , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Hipertensão Pulmonar/fisiopatologia , Leucócitos Mononucleares , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
RATIONALE: Idiopathic pulmonary fibrosis (IPF) involves the accumulation of α-smooth muscle actin-expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor-ß1 (TGF-ß1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF. OBJECTIVES: We aimed to define an association between mtDNA and fibroblast responses in IPF. METHODS: We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF-ß1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects. MEASUREMENTS AND MAIN RESULTS: Exposure to mtDNA augments α-smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF-ß1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event-free survival. CONCLUSIONS: These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.
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DNA Mitocondrial/metabolismo , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/mortalidade , Idoso , Intervalo Livre de Doença , Feminino , Humanos , MasculinoRESUMO
Pulmonary fibrosis is a progressive and often fatal condition that is believed to be partially orchestrated by macrophages. Mechanisms that control migration of these cells into and within the lung remain undefined. We evaluated the contributions of the semaphorin receptor, plexin C1 (PLXNC1), and the exocytic calcium sensor, synaptotagmin 7 (Syt7), in these processes. We evaluated the role of PLXNC1 in macrophage migration by using Boyden chambers and scratch tests, characterized its contribution to experimentally induced lung fibrosis in mice, and defined the mechanism for our observations. Our findings reveal that relative to control participants, patients with idiopathic pulmonary fibrosis demonstrate excessive monocyte migration and underexpression of PLXNC1 in the lungs and circulation, a finding that is recapitulated in the setting of scleroderma-related interstitial lung disease. Relative to wild type, PLXNC1-/- mouse macrophages are excessively migratory, and PLXNC1-/- mice show exacerbated collagen accumulation in response to either inhaled bleomycin or inducible lung targeted TGF-ß1 overexpression. These findings are ameliorated by replacement of PLXNC1 on bone marrow-derived cells or by genetic deletion of Syt7. These data demonstrate the previously unrecognized observation that PLXNC1 deficiency permits Syt7-mediated macrophage migration and enhances mammalian lung fibrosis.-Peng, X., Moore, M., Mathur, A., Zhou, Y., Sun, H., Gan, Y., Herazo-Maya, J. D., Kaminski, N., Hu, X., Pan, H., Ryu, C., Osafo-Addo, A., Homer, R. J., Feghali-Bostwick, C., Fares, W. H., Gulati, M., Hu, B., Lee, C.-G., Elias, J. A., Herzog, E. L. Plexin C1 deficiency permits synaptotagmin 7-mediated macrophage migration and enhances mammalian lung fibrosis.
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Macrófagos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fibrose Pulmonar/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Sinaptotagminas/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Pulmão/metabolismo , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Fibrose Pulmonar/genética , Receptores de Superfície Celular/deficiência , Receptores Virais/deficiência , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis and variable clinical course. Although matrix metalloproteinase-7 (MMP-7) is emerging as an important IPF biomarker, reproducibility across studies is unclear. We aimed to determine whether a previously reported prognostic threshold for MMP-7 was predictive of mortality in an independent cohort of IPF patients. METHODS: MMP-7 concentrations obtained from heparinized plasma samples were determined by ELISA in 97 patients with IPF and 41 healthy controls. The association of the previously published heparin plasma MMP-7 threshold of 12.1 ng/mL with all-cause mortality or transplant-free survival (TFS) was determined, either as an independent biomarker or as part of the modified personal clinical and molecular mortality index (m-PCMI). RESULTS: MMP-7 plasma concentrations were significantly higher in IPF patients compared to healthy controls (14.40 ± 6.55 ng/mL vs 6.03 ± 2.51 ng/mL, P < 0.001). The plasma MMP-7 threshold of 12.1 ng/mL was significantly associated with both all-cause mortality and TFS (unadjusted Cox proportional hazard ratio (HR) = 25.85 and 15.49, 95% CI: 10.91-61.23 and 5.41-44.34, respectively, P < 0.001). MMP-7 concentrations, split by 12.1 ng/mL, were significantly (P < 0.05) predictive of mortality and TFS after adjusting for age, gender, smoking and baseline pulmonary function parameters, in a multivariate Cox proportional hazards model. MMP-7 concentrations were negatively correlated with diffusing lung capacity of carbon monoxide (DLCO ) (r = -0.21, P = 0.02), and positively with a mortality risk scoring system (GAP) that combines age, gender, forced vital capacity (FVC) and DLCO (r = 0.32, P = 0.001). CONCLUSION: This study confirms that MMP-7 concentrations could be used to accurately predict outcomes across cohorts and centres, when similar collection protocols are applied.
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Fibrose Pulmonar Idiopática/sangue , Fibrose Pulmonar Idiopática/mortalidade , Metaloproteinase 7 da Matriz/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Fibrose Pulmonar Idiopática/fisiopatologia , Fibrose Pulmonar Idiopática/cirurgia , Transplante de Pulmão , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Capacidade de Difusão Pulmonar , Reprodutibilidade dos Testes , Taxa de SobrevidaAssuntos
Fibrose Pulmonar Idiopática , Metformina , Bleomicina , Humanos , Hidroxicloroquina , Hormônios TireóideosRESUMO
BACKGROUND: Dysregulation of the airway microbiota is thought to contribute to airway inflammation in both chronic rhinosinusitis (CRS) and asthma. However, the relationship between the upper and lower airway microbiome remains unclear. METHODS: Sinus and lung brushes were collected from 29 CRS participants undergoing sinus surgery. DNA was extracted and submitted for 16s rRNA microbiome sequencing. Alpha and beta diversity metrics, taxonomic composition, and differences between individual taxa were compared for paired sinus and bronchial samples. RESULTS: Twenty-three out of 29 participants had sufficient samples for analysis. The mean (standard deviation) age was 51.59 (14.57) years, and 10 (44%) patients were female. Twelve (52%) patients had comorbid asthma. Sinus brushes had significantly higher alpha diversity indexes (Shannon and Faith) compared to bronchial brushes (p < 0.001). Beta diversity metrics were significantly different between the sinus and bronchial samples. Principal coordinate analysis showed no clustering of paired nasal and bronchial samples. Sinus brushes had significantly more Lawsonella, Corynebacterium, and Staphylococcus compared to bronchia brushes, while the latter were enriched in Tropheryma and Sphingomonas, among others (false discovery rate [FDR]-adjusted p < 0.01). Finally, CRS patients with comorbid asthma had significantly higher Pseudomonas and Peptoniphilus in sinus brushes and lower Prevotella in bronchial brushes when compared to non-asthmatics (FDR-adjusted p < 0.01). CONCLUSION: The sinus and bronchial bacterial microbiomes differ in important ways. Our study suggests that migration of bacteria from the sinus into the lower airways is unlikely in patients with CRS.
Assuntos
Asma , Microbiota , Seios Paranasais , Rinite , Sinusite , Humanos , Sinusite/microbiologia , Feminino , Rinite/microbiologia , Pessoa de Meia-Idade , Masculino , Doença Crônica , Adulto , Asma/microbiologia , Seios Paranasais/microbiologia , Idoso , Brônquios/microbiologia , RNA Ribossômico 16S/genética , Bactérias/isolamento & purificação , Bactérias/genética , Bactérias/classificação , Pulmão/microbiologia , RinossinusiteRESUMO
Host response aimed at eliminating the infecting pathogen, as well as the pathogen itself, can cause tissue injury. Tissue injury leads to the release of a myriad of cellular components including mitochondrial DNA, which the host senses through pattern recognition receptors. How the sensing of tissue injury by the host shapes the anti-pathogen response remains poorly understood. In this study, we utilized mice that are deficient in toll-like receptor-9 (TLR9), which binds to unmethylated CpG DNA sequences such as those present in bacterial and mitochondrial DNA. To avoid direct pathogen sensing by TLR9, we utilized the influenza virus, which lacks ligands for TLR9, to determine how damage sensing by TLR9 contributes to anti-influenza immunity. Our data show that TLR9-mediated sensing of tissue damage promotes an inflammatory response during early infection, driven by the myeloid cells and associated cytokine responses. Along with the diminished inflammatory response, the absence of damage sensing through TLR9 led to impaired viral clearance manifested as a higher and prolonged influenza burden in the lung. The absence of TLR9 led to extensive infection of myeloid cells including monocytes and macrophages rendering them highly inflammatory, despite having a low initial inflammatory response. The persistent inflammation driven by infected myeloid cells led to persistent lung injury and impaired recovery in influenza-infected TLR9-/- mice. Further, we show elevated circulating TLR9 ligands in the plasma samples of patients with influenza, demonstrating its clinical relevance. Overall, over data show an essential role of damage sensing through TLR9 in promoting anti-influenza immunity.