RESUMO
Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.
Assuntos
Macrófagos , Rhinovirus , Humanos , Macrófagos/microbiologia , Macrófagos Alveolares , Fagocitose , BactériasRESUMO
AIMS: Corticosteroids are the treatment of choice for many inflammatory diseases but often lead to adverse effects, including hyperglycaemia. This study investigated the mechanisms driving differential effects on glucose control for AZD9567, an oral nonsteroidal selective glucocorticoid receptor modulator vs. prednisolone in 46 patients with type 2 diabetes mellitus. METHODS: In this randomized, double-blind, 2-way cross-over study (NCT04556760), participants received either AZD9567 72 mg and prednisolone 40 mg daily (cohort 1); AZD9567 40 mg and prednisolone 20 mg daily (cohort 2); or placebo and prednisolone 5 mg daily (cohort 3). Treatment duration was 3 days with a 3-week washout between treatment periods. Glycaemic control was assessed after a standardized meal and with continuous glucose monitoring. RESULTS: A significant difference between AZD9567 and prednisolone in favour of AZD9567 was observed for the change from baseline to Day 4 glucose excursions postmeal in cohort 1 (glucose area under the curve from 0 to 4 h -4.54%; 95% confidence interval [CI]: -8.88, -0.01; P = .049), but not in cohort 2 (-5.77%; 95% CI: -20.92, 12.29; P = .435). In cohort 1, significant differences between AZD9567 and prednisolone were also seen for the change from baseline to day 4 in insulin and glucagon secretion postmeal (P < .001 and P = .005, respectively) and change from baseline to Day 4 in GLP-1 response (P = .022). Significant differences between AZD9567 and prednisolone for 24-h glucose control were observed for both cohort 1 (-1.507 mmol/L; 95% CI: -2.0820, -0.9314; P < .001) and cohort 2 (-1.110 mmol/L; 95% CI -1.7257, -0.4941; P < .001). CONCLUSION: AZD9567 significantly reduced treatment-induced hyperglycaemia compared with prednisolone.
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Glicemia , Estudos Cross-Over , Diabetes Mellitus Tipo 2 , Controle Glicêmico , Prednisolona , Humanos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/sangue , Masculino , Pessoa de Meia-Idade , Prednisolona/administração & dosagem , Prednisolona/uso terapêutico , Prednisolona/efeitos adversos , Feminino , Método Duplo-Cego , Glicemia/efeitos dos fármacos , Idoso , Hiperglicemia/induzido quimicamente , Hiperglicemia/sangue , Hiperglicemia/tratamento farmacológico , Glucocorticoides/administração & dosagem , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacologia , Adulto , Insulina , Receptores de Glucocorticoides , Período Pós-PrandialRESUMO
Human rhinovirus is a causative agent of severe exacerbations of chronic obstructive pulmonary disease (COPD). COPD is characterised by an increased number of alveolar macrophages with diminished phagocytic functions, but how rhinovirus infection affects macrophage function is still unknown. Here, we describe that human rhinovirus 16 impairs bacterial uptake and receptor-mediated phagocytosis in macrophages. The stalled phagocytic cups contain accumulated F-actin. Interestingly, we find that human rhinovirus 16 downregulates the expression of Arpin, a negative regulator of the Arp2/3 complex. Importantly, re-expression of the protein rescues defective internalisation in human rhinovirus 16-treated cells, demonstrating that Arpin is a key factor targeted to impair phagocytosis. We further show that Arpin is required for efficient uptake of multiple targets, for F-actin cup formation and for successful phagosome completion in macrophages. Interestingly, Arpin is recruited to sites of membrane extension and phagosome closure. Thus, we identify Arpin as a central actin regulator during phagocytosis that it is targeted by human rhinovirus 16, allowing the virus to perturb bacterial internalisation and phagocytosis in macrophages.
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Fagocitose , Rhinovirus , Proteínas de Transporte , Humanos , Macrófagos , Macrófagos Alveolares , FagossomosRESUMO
Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation, small airway remodeling, and emphysema. Airway remodeling in patients with COPD involves both the airway epithelium and the subepithelial extracellular matrix (ECM). However, it is currently unknown how epithelial remodeling in COPD airways depends on the relative influence from inherent defects in the epithelial cells and alterations in the ECM. To address this, we analyzed global gene expression in COPD human bronchial epithelial cells (HBEC) and normal HBEC after repopulation on decellularized bronchial scaffolds derived from patients with COPD or donors without COPD. COPD HBEC grown on bronchial scaffolds showed an impaired ability to initiate ciliated-cell differentiation, which was evident on all scaffolds regardless of their origin. In addition, although normal HBEC were less affected by the disease state of the bronchial scaffolds, COPD HBEC showed a gene expression pattern indicating increased proliferation and a retained basal-cell phenotype when grown on COPD bronchial scaffolds compared with normal bronchial scaffolds. By using mass spectrometry, we identified 13 matrisome proteins as being differentially abundant between COPD bronchial scaffolds and normal bronchial scaffolds. These observations are consistent with COPD pathology and suggest that both epithelial cells and the ECM contribute to epithelial-cell remodeling in COPD airways.
Assuntos
Brônquios/química , Diferenciação Celular , Células Epiteliais/metabolismo , Matriz Extracelular/química , Doença Pulmonar Obstrutiva Crônica/metabolismo , Alicerces Teciduais/química , Células Epiteliais/patologia , Feminino , Humanos , Masculino , Doença Pulmonar Obstrutiva Crônica/patologiaRESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) patients are at increased risk of poor outcome from Coronavirus disease (COVID-19). Early data suggest elevated Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) receptor angiotensin converting enzyme 2 (ACE2) expression, but relationships to disease phenotype and downstream regulators of inflammation in the Renin-Angiotensin system (RAS) are unknown. We aimed to determine the relationship between RAS gene expression relevant to SARS-CoV-2 infection in the lung with disease characteristics in COPD, and the regulation of newly identified SARS-CoV-2 receptors and spike-cleaving proteases, important for SARS-CoV-2 infection. METHODS: We quantified gene expression using RNA sequencing of epithelial brushings and bronchial biopsies from 31 COPD and 37 control subjects. RESULTS: ACE2 gene expression (log2-fold change (FC)) was increased in COPD compared to ex-smoking (HV-ES) controls in epithelial brushings (0.25, p = 0.042) and bronchial biopsies (0.23, p = 0.050), and correlated with worse lung function (r = - 0.28, p = 0.0090). ACE2 was further increased in frequent exacerbators compared to infrequent exacerbators (0.51, p = 0.00045) and associated with use of ACE inhibitors (ACEi) (0.50, p = 0.0034), having cardiovascular disease (0.23, p = 0.048) or hypertension (0.34, p = 0.0089), and inhaled corticosteroid use in COPD subjects in bronchial biopsies (0.33, p = 0.049). Angiotensin II receptor type (AGTR)1 and 2 expression was decreased in COPD bronchial biopsies compared to HV-ES controls with log2FC of -0.26 (p = 0.033) and - 0.40, (p = 0.0010), respectively. However, the AGTR1:2 ratio was increased in COPD subjects compared with HV-ES controls, log2FC of 0.57 (p = 0.0051). Basigin, a newly identified potential SARS-CoV-2 receptor was also upregulated in both brushes, log2FC of 0.17 (p = 0.0040), and bronchial biopsies, (log2FC of 0.18 (p = 0.017), in COPD vs HV-ES. Transmembrane protease, serine (TMPRSS)2 was not differentially regulated between control and COPD. However, various other spike-cleaving proteases were, including TMPRSS4 and Cathepsin B, in both epithelial brushes (log2FC of 0.25 (p = 0.0012) and log2FC of 0.56 (p = 5.49E-06), respectively) and bronchial biopsies (log2FC of 0.49 (p = 0.00021) and log2FC of 0.246 (p = 0.028), respectively). CONCLUSION: This study identifies key differences in expression of genes related to susceptibility and aetiology of COVID-19 within the COPD lung. Further studies to understand the impact on clinical course of disease are now required.
Assuntos
COVID-19/genética , Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Transcriptoma , Idoso , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Basigina/genética , Basigina/metabolismo , COVID-19/diagnóstico , COVID-19/metabolismo , COVID-19/fisiopatologia , Estudos de Casos e Controles , Feminino , Volume Expiratório Forçado , Regulação da Expressão Gênica , Humanos , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Capacidade VitalRESUMO
Asthma exacerbations are triggered by rhinovirus infections. We employed a systems biology approach to delineate upper-airway gene network patterns underlying asthma exacerbation phenotypes in children. Cluster analysis unveiled distinct IRF7hi versus IRF7lo molecular phenotypes, the former exhibiting robust upregulation of Th1/type I IFN responses and the latter an alternative signature marked by upregulation of cytokine and growth factor signaling and downregulation of IFN-γ. The two phenotypes also produced distinct clinical phenotypes. For IRF7lo children, symptom duration prior to hospital presentation was more than twice as long from initial symptoms (p = 0.011) and nearly three times as long for cough (p < 0.001), the odds ratio of admission to hospital was increased more than 4-fold (p = 0.018), and time to recurrence was shorter (p = 0.015). In summary, our findings demonstrate that asthma exacerbations in children can be divided into IRF7hi versus IRF7lo phenotypes with associated differences in clinical phenotypes.
Assuntos
Asma/genética , Fator Regulador 7 de Interferon/genética , Sons Respiratórios/genética , Infecções Respiratórias , Adolescente , Asma/imunologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Análise por Conglomerados , Feminino , Redes Reguladoras de Genes , Humanos , Lactente , Recém-Nascido , Masculino , Fenótipo , Sons Respiratórios/imunologia , Infecções Respiratórias/complicações , Infecções Respiratórias/genética , Infecções Respiratórias/imunologia , TranscriptomaRESUMO
BACKGROUND: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear. OBJECTIVE: We sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients. METHODS: An IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens. RESULTS: Activation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS-high asthma with increased epithelial expression of IL-6TS-inducible genes in the absence of systemic inflammation. The IL-6TS-high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1ß, IL-8, and IL-1ß. CONCLUSIONS: Local lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.
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Asma/imunologia , Biomarcadores/metabolismo , Células Epiteliais/fisiologia , Inflamação/imunologia , Interleucina-6/metabolismo , Pulmão/fisiologia , Escarro/metabolismo , Adulto , Remodelação das Vias Aéreas , Células Cultivadas , Estudos de Coortes , Estudos Transversais , Regulação da Expressão Gênica , Humanos , Masculino , Fenótipo , Receptores de Interleucina-6/metabolismo , Hipersensibilidade Respiratória , Transdução de Sinais , TranscriptomaRESUMO
OBJECTIVES: Genetic variations in TNFAIP3 (A20) de-ubiquitinase (DUB) domain increase the risk of systemic lupus erythematosus (SLE) and rheumatoid arthritis. A20 is a negative regulator of NF-κB but the role of its DUB domain and related genetic variants remain unclear. We aimed to study the functional effects of A20 DUB-domain alterations in immune cells and understand its link to SLE pathogenesis. METHODS: CRISPR/Cas9 was used to generate human U937 monocytes with A20 DUB-inactivating C103A knock-in (KI) mutation. Whole genome RNA-sequencing was used to identify differentially expressed genes between WT and C103A KI cells. Functional studies were performed in A20 C103A U937 cells and in immune cells from A20 C103A mice and genotyped healthy individuals with A20 DUB polymorphism rs2230926. Neutrophil extracellular trap (NET) formation was addressed ex vivo in neutrophils from A20 C103A mice and SLE-patients with rs2230926. RESULTS: Genetic disruption of A20 DUB domain in human and murine myeloid cells did not give rise to enhanced NF-κB signalling. Instead, cells with C103A mutation or rs2230926 polymorphism presented an upregulated expression of PADI4, an enzyme regulating protein citrullination and NET formation, two key mechanisms in autoimmune pathology. A20 C103A cells exhibited enhanced protein citrullination and extracellular trap formation, which could be suppressed by selective PAD4 inhibition. Moreover, SLE-patients with rs2230926 showed increased NETs and increased frequency of autoantibodies to citrullinated epitopes. CONCLUSIONS: We propose that genetic alterations disrupting the A20 DUB domain mediate increased susceptibility to SLE through the upregulation of PADI4 with resultant protein citrullination and extracellular trap formation.
Assuntos
Citrulinação/genética , Endopeptidases/genética , Armadilhas Extracelulares/genética , Lúpus Eritematoso Sistêmico/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Epitopos/imunologia , Predisposição Genética para Doença/genética , Humanos , Lúpus Eritematoso Sistêmico/sangue , Camundongos , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Polimorfismo Genético , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Regulação para Cima/genéticaRESUMO
Background: Janus Kinase (JAK) inhibition has recently demonstrated therapeutic efficacy in both restoring hair growth and resolving inflammation in Alopecia Areata (AA). These effects are dose dependent and mainly efficacious at ranges close to a questionable risk profile. Objectives: We explored the possibility to separate the beneficial and adverse effects of JAK inhibition by selectively inhibiting JAK1 and thereby avoiding side effects associated with JAK2 blockade. Methods: The C3H/HeJ mouse model of AA was used to demonstrate therapeutic efficacy in vivo with different regimens of a selection of JAK inhibitors in regards to systemic versus local drug exposure. Human peripheral blood lymphocytes were stimulated in vitro to demonstrate translation to the human situation. Results: We demonstrate that selective inhibition of JAK1 produces fast resolution of inflammation and complete restoration of hair growth in the C3H/HeJ mouse model of AA. Furthermore, we show that topical treatment does not restore hair growth and that treatment needs to be extended well beyond that of restored hair growth in order to reach treatment-free remission. For translatability to human disease, we show that cytokines involved in AA pathogenesis are similarly inhibited by selective JAK1 and pan-JAK inhibition in stimulated human peripheral lymphocytes and specifically in CD8+ T cells. Conclusion: This study demonstrates that systemic exposure is required for efficacy in AA and we propose that a selective JAK1 inhibitor will offer a treatment option with a superior safety profile to pan-JAK inhibitors for these patients.
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Oral corticosteroid use is limited by side effects, some caused by off-target actions on the mineralocorticoid receptor that disrupt electrolyte balance. AZD9567 is a selective, nonsteroidal glucocorticoid receptor modulator. The efficacy, safety, and tolerability of AZD9567 and prednisolone were assessed in a phase IIa study. Anti-inflammatory mechanism of action was also evaluated in vitro in monocytes from healthy donors. In this randomized, double-blind, parallel-group, multicenter study, patients with active rheumatoid arthritis were randomized 1:1 to AZD9567 40 mg or prednisolone 20 mg once daily orally for 14 days. The primary end point was change from baseline in DAS28-CRP at day 15. Secondary end points included components of DAS28-CRP, American College of Rheumatology (ACR) response criteria (ACR20, ACR50, and ACR70), and safety end points, including serum electrolytes. Overall, 21 patients were randomized to AZD9567 (n = 11) or prednisolone (n = 10), and all completed the study. As anticipated, AZD9567 had a similar efficacy profile to prednisolone, with no clinically meaningful (i.e., >1.0) difference in change from baseline to day 15 in DAS28-CRP between AZD9567 and prednisolone (least-squares mean difference: 0.47, 95% confidence interval: -0.49 to 1.43). Similar results were observed for the secondary efficacy end points. In vitro transcriptomic analysis showed that anti-inflammatory responses were similar for AZD9567, prednisolone, and dexamethasone. Unlike prednisolone, AZD9567 had no effect on the serum sodium:potassium ratio. The safety profile was not different from that of prednisolone. Larger studies of longer duration are required to determine whether AZD9567 40 mg may in the future be an alternative to prednisolone in patients with inflammatory disease.
Assuntos
Antirreumáticos , Artrite Reumatoide , Humanos , Prednisolona/efeitos adversos , Antirreumáticos/uso terapêutico , Resultado do Tratamento , Artrite Reumatoide/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Método Duplo-Cego , Metotrexato/uso terapêuticoRESUMO
Rationale: Pulmonary surfactant is vital for lung homeostasis as it reduces surface tension to prevent alveolar collapse and provides essential immune-regulatory and antipathogenic functions. Previous studies demonstrated dysregulation of some individual surfactant components in COPD. We investigated relationships between COPD disease measures and dysregulation of surfactant components to gain new insights into potential disease mechanisms. Methods: Bronchoalveolar lavage proteome and lipidome were characterised in ex-smoking mild/moderate COPD subjects (n=26) and healthy ex-smoking (n=20) and never-smoking (n=16) controls using mass spectrometry. Serum surfactant protein analysis was performed. Results: Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, surfactant protein (SP)-B, SP-A and SP-D concentrations were lower in COPD versus controls (log2 fold change (log2FC) -2.0, -2.2, -1.5, -0.5, -0.7 and -0.5 (adjusted p<0.02), respectively) and correlated with lung function. Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D, napsin A and CD44 inversely correlated with computed tomography small airways disease measures (expiratory to inspiratory mean lung density) (r= -0.56, r= -0.58, r= -0.45, r= -0.36, r= -0.44, r= -0.37, r= -0.40 and r= -0.39 (adjusted p<0.05)). Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D and NAPSA inversely correlated with emphysema (% low-attenuation areas): r= -0.55, r= -0.61, r= -0.48, r= -0.51, r= -0.41, r= -0.31 and r= -0.34, respectively (adjusted p<0.05). Neutrophil elastase, known to degrade SP-A and SP-D, was elevated in COPD versus controls (log2FC 0.40, adjusted p=0.0390), and inversely correlated with SP-A and SP-D. Serum SP-D was increased in COPD versus healthy ex-smoking volunteers, and predicted COPD status (area under the curve 0.85). Conclusions: Using a multiomics approach, we demonstrate, for the first time, global surfactant dysregulation in COPD that was associated with emphysema, giving new insights into potential mechanisms underlying the cause or consequence of disease.
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BACKGROUND: At the end of the 19th century, infant mortality was high in urban and rural areas in Sweden. In Stockholm, the mortality rate was particularly high among foster children. This study addresses the importance for health of targeted public policies and their local implementation in the reduction of excess mortality among foster children in Stockholm at the turn of the 19th century. In response to public concern, a law was passed in 1902 on inspections of foster homes. Stockholm city employed a handful of inspectors who visited foster homes and advised parents on child care and feeding. METHODS: Analysis of historical records from the City of Stockholm was combined with epidemiological analysis of mortality rates and hazard ratios on individual-level data for 112, 746 children aged <1 year residing in one part of Stockholm between 1878 and 1925. Hazard ratios of mortality were calculated using Cox' regression analysis. RESULTS: Mortality rates of foster infants exceeded 300/1000 before 1903. Ten years later the mortality rates among foster children had declined and were similar to other children born in and out of wedlock. Historical accounts and epidemiological analysis of individual-level data over a longer time period showed similar results. CONCLUSIONS: Targeted policy measures to foster children may have potentiated the positive health effects of other universal policies, such as improved living conditions, clean water and sanitation for the whole population in the city, contributing to an equalization of mortality rates between different groups.
Assuntos
Cuidados no Lar de Adoção/história , Cuidados no Lar de Adoção/legislação & jurisprudência , Mortalidade Infantil/história , Política Pública/história , Análise de Sobrevida , Cidades/epidemiologia , História do Século XIX , História do Século XX , Humanos , Lactente , Mortalidade Infantil/tendências , Modelos de Riscos Proporcionais , Sistema de Registros , Suécia/epidemiologiaRESUMO
Iron is a key element for systemic oxygen delivery and cellular energy metabolism. Thus regulation of systemic and local iron metabolism is key for maintaining energy homeostasis. Significant changes in iron levels due to malnutrition or hemorrhage, have been associated with several diseases such as hemochromatosis, liver cirrhosis and COPD. Macrophages are key cells in regulating iron levels in tissues as they sequester excess iron. How iron overload affects macrophage differentiation and function remains a subject of debate. Here we used an in vitro model of monocyte-to-macrophage differentiation to study the effect of iron overload on macrophage function. We found that providing excess iron as soluble ferric ammonium citrate (FAC) rather than as heme-iron complexes derived from stressed red blood cells (sRBC) interferes with macrophage differentiation and phagocytosis. Impaired macrophage differentiation coincided with increased expression of oxidative stress-related genes. Addition of FAC also led to increased levels of cellular and mitochondrial reactive oxygen species (ROS) and interfered with mitochondrial function and ATP generation. The effects of iron overload were reproduced by the mitochondrial ROS-inducer rotenone while treatment with the ROS-scavenger N-Acetylcysteine partially reversed FAC-induced effects. Finally, we found that iron-induced oxidative stress interfered with upregulation of M-CSFR and MAFB, two crucial determinants of macrophage differentiation and function. In summary, our findings suggest that high levels of non-heme iron interfere with macrophage differentiation by inducing mitochondrial oxidative stress. These findings might be important to consider in the context of diseases like chronic obstructive pulmonary disease (COPD) where both iron overload and defective macrophage function have been suggested to play a role in disease pathogenesis.
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Sobrecarga de Ferro , Doença Pulmonar Obstrutiva Crônica , Humanos , Espécies Reativas de Oxigênio/metabolismo , Monócitos/metabolismo , Sobrecarga de Ferro/metabolismo , Estresse Oxidativo , Ferro/metabolismo , Macrófagos/metabolismoRESUMO
Plant mitogenomes can be difficult to assemble because they are structurally dynamic and prone to intergenomic DNA transfers, leading to the unusual situation where an organelle genome is far outnumbered by its nuclear counterparts. As a result, comparative mitogenome studies are in their infancy and some key aspects of genome evolution are still known mainly from pregenomic, qualitative methods. To help address these limitations, we combined machine learning and in silico enrichment of mitochondrial-like long reads to assemble the bacterial-sized mitogenome of Norway spruce (Pinaceae: Picea abies). We conducted comparative analyses of repeat abundance, intergenomic transfers, substitution and rearrangement rates, and estimated repeat-by-repeat homologous recombination rates. Prompted by our discovery of highly recombinogenic small repeats in P. abies, we assessed the genomic support for the prevailing hypothesis that intramolecular recombination is predominantly driven by repeat length, with larger repeats facilitating DNA exchange more readily. Overall, we found mixed support for this view: Recombination dynamics were heterogeneous across vascular plants and highly active small repeats (ca. 200 bp) were present in about one-third of studied mitogenomes. As in previous studies, we did not observe any robust relationships among commonly studied genome attributes, but we identify variation in recombination rates as a underinvestigated source of plant mitogenome diversity.
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Genoma Mitocondrial , Picea/genética , Recombinação Genética , Simulação por Computador , Cycadopsida/genética , DNA de Plantas/química , Genes de Plantas , Variação Genética , Sequências Repetitivas de Ácido Nucleico , Máquina de Vetores de SuporteRESUMO
Background: Unlike p38 mitogen-activated protein Kinases (MAPK) that has been extensively studied in the context of lung-associated pathologies in COPD, the role of the dual-specificity mitogen-activated protein kinase kinase (MEK1/2) or its downstream signaling molecule extracellular signal-regulated kinases 1/2 (ERK1/2) in COPD is poorly understood. Objectives: The aim of this study was to address whether MEK1/2 pathway activation is linked to COPD and that targeting this pathway can improve lung inflammation through decreased immune-mediated inflammatory responses without compromising bacterial clearance. Methods: Association of MEK1/2 pathway activation to COPD was investigated by immunohistochemistry using lung tissue biopsies from COPD and healthy individuals and through analysis of sputum gene expression data from COPD patients. The anti-inflammatory effect of MEK1/2 inhibition was assessed on cytokine release from lipopolysaccharide-stimulated alveolar macrophages. The effect of MEK1/2 inhibition on bacterial clearance was assessed using Staphylococcus aureus killing assays with RAW 264.7 macrophage cell line and human neutrophils. Results: We report here MEK1/2 pathway activation demonstrated by increased pERK1/2 staining in bronchial epithelium and by the presence of MEK gene activation signature in sputum samples from COPD patients. Inhibition of MEK1/2 resulted in a superior anti-inflammatory effect in human alveolar macrophages in comparison to a p38 inhibitor. Furthermore, MEK1/2 inhibition led to an increase in bacterial killing in human neutrophils and RAW 264.7 cells that was not observed with the p38 inhibitor. Conclusion: Our data demonstrate the activation of MEK1/2 pathway in COPD and highlight a dual function of MEK1/2 inhibition in improving host defense responses whilst also controlling inflammation.
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Benzamidas/farmacologia , Benzamidas/uso terapêutico , Difenilamina/análogos & derivados , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Adulto , Idoso , Células Cultivadas , Difenilamina/farmacologia , Difenilamina/uso terapêutico , Feminino , Humanos , Inflamação/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Transdução de Sinais/efeitos dos fármacos , Adulto JovemRESUMO
Chronic obstructive pulmonary disease (COPD) is a serious global health problem characterized by chronic airway inflammation, progressive airflow limitation and destruction of lung parenchyma. Remodeling of the bronchial airways in COPD includes changes in both the bronchial epithelium and the subepithelial extracellular matrix (ECM). To explore the impact of an aberrant ECM on epithelial cell phenotype in COPD we developed a new ex vivo model, in which normal human bronchial epithelial (NHBE) cells repopulate and differentiate on decellularized human bronchial scaffolds derived from COPD patients and healthy individuals. By using transcriptomics, we show that bronchial ECM from COPD patients induces differential gene expression in primary NHBE cells when compared to normal bronchial ECM. The gene expression profile indicated altered activity of upstream mediators associated with COPD pathophysiology, including hepatocyte growth factor, transforming growth factor beta 1 and platelet-derived growth factor B, which suggests that COPD-related changes in the bronchial ECM contribute to the defective regenerative ability in the airways of COPD patients.
Assuntos
Matriz Extracelular/genética , Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Transcriptoma/genética , Brônquios/metabolismo , Brônquios/patologia , Fator de Crescimento do Tecido Conjuntivo/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Matriz Extracelular/patologia , Regulação da Expressão Gênica/genética , Fator de Crescimento de Hepatócito/genética , Humanos , Pulmão/patologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-sis/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Fator de Crescimento Transformador beta1/genéticaRESUMO
Nitric oxide (NO·) is a messenger molecule with diverse physiological roles including host defense, neurotransmission and vascular function. The synthesis of NO· from l-arginine is catalyzed by NO-synthases and occurs in two steps through the intermediary Nω-hydroxy-l-arginine (NHA). In both steps the P450-like reaction cycle is coupled with the redox cycle of the cofactor tetrahydrobiopterin (H4B). The mechanism of the second step is studied by Density Functional Theory calculations to ascertain the canonical sequence of proton and electron transfer (PT and ET) events. The proposed mechanism is controlled by the interplay of two electron donors, H4B and NHA. Consistent with experimental data, the catalytic cycle proceeds through the ferric-hydroperoxide complex (Cpd 0) and the following aqua-ferriheme resting state, and involves interim partial oxidation of H4B. The mechanism starts with formation of Cpd 0 from the ferrous-dioxy reactant complex by PT from the C-ring heme propionate coupled with hole transfer to H4B through the highest occupied π-orbital of NHA as a bridge. This enables PT from NHA+· to the proximal oxygen leading to the shallow ferriheme-H2O2 oxidant. Subsequent Fenton-like peroxide bond cleavage triggered by ET from the NHA-derived iminoxy-radical leads to the protonated Cpd II diradicaloid singlet stabilized by spin delocalization in H4B, and the closed-shell coordination complex of HO- with iminoxy-cation. The complex is converted to the transient C-adduct, which releases intended products upon PT to the ferriheme-HO- complex coupled with ET to the H4B+·. Deferred ET from the substrate or undue ET from/to the cofactor leads to side products.
Assuntos
Arginina/análogos & derivados , Biopterinas/análogos & derivados , Modelos Moleculares , NADP/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Arginina/química , Arginina/metabolismo , Biocatálise , Biopterinas/química , Biopterinas/metabolismo , Domínio Catalítico , Citrulina/química , Citrulina/metabolismo , Sequência Conservada , Bases de Dados de Proteínas , Transporte de Elétrons , Humanos , Ligação de Hidrogênio , NADP/química , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/química , Oxirredução , Prótons , Teoria Quântica , TermodinâmicaRESUMO
BACKGROUND: BET proteins (BRD2, BRD3, BRDT and BRD4) belong to the family of bromodomain containing proteins, which form a class of transcriptional co-regulators. BET proteins bind to acetylated lysine residues in the histones of nucleosomal chromatin and function either as co-activators or co-repressors of gene expression. An imbalance between HAT and HDAC activities resulting in hyperacetylation of histones has been identified in COPD. We hypothesized that pan-BET inhibitor (JQ1) treatment of BET protein interactions with hyperacetylated sites in the chromatin will regulate excessive activation of pro-inflammatory genes in key inflammatory drivers of alveolar macrophages (AM) in COPD. METHODS AND FINDINGS: Transcriptome analysis of AM from COPD patients indicated up-regulation of macrophage M1 type genes upon LPS stimulation. Pan-BET inhibitor JQ1 treatment attenuated expression of multiple genes, including pro-inflammatory cytokines and regulators of innate and adaptive immune cells. We demonstrated for the first time that JQ1 differentially modulated LPS-induced cytokine release from AM or peripheral blood mononuclear cells (PBMC) of COPD patients compared to PBMC of healthy controls. Using the BET regulated gene signature, we identified a subset of COPD patients, which we propose to benefit from BET inhibition. CONCLUSIONS: This work demonstrates that the effects of pan-BET inhibition through JQ1 treatment of inflammatory cells differs between COPD patients and healthy controls, and the expression of BET protein regulated genes is altered in COPD. These findings provide evidence of histone hyperacetylation as a mechanism driving chronic inflammatory changes in COPD.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fatores de Transcrição/metabolismo , Azepinas/farmacologia , Estudos de Casos e Controles , Proteínas de Ciclo Celular , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Humanos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Doença Pulmonar Obstrutiva Crônica/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Triazóis/farmacologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0173115.].
RESUMO
As in European countries a century ago, diarrhea is a major cause of child mortality in poor countries today. In Stockholm at the turn of the 19th century, political commitment, infrastructural investments in water and sanitation, and enforcement of sanitary improvements by a strong implementing organization helped eliminate diarrhea as a principal cause of death among children. These interventions also had an equitable impact on social class differences in diarrhea mortality, but not on overall mortality; overall mortality declined, but class differences remained. General infrastructural improvement and health education coupled with targeted interventions to vulnerable children may be successful in improving child health and reducing social differentials in mortality. Specific health care interventions may need to be complemented by infrastructural investments to improve water and sanitation if diarrhea mortality is to be further reduced in poor countries today.