Multiomics links global surfactant dysregulation with airflow obstruction and emphysema in COPD

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

Functional characterisation and role of Tissue Transglutaminase during the progression of colorectal cancer

Colorectal cancer (CRC) is the 4th most lethal cancer worldwide. Currently available chemotherapy treatments used in combination with surgery have toxic effects which limit their prolonged usage. Thus, there is still great need for less toxic and more specific treatments, alongside better predictive and prognostic disease markers. Tissue Transglutaminase (TG2) is a multi-functional enzyme whose role in cancer can be either tumour-promoting or tumour-suppressing depending on cell type and intracellular localisation. There is a large body of literature dissecting TG2 tumour-promoting role, whereas very little is known about its tumour-suppressing functions. MicroRNAs are small RNA molecules with translation regulation functions. Their altered expression in cancer causes abnormal translation of their target mRNAs. The aim of this work was to fully characterise TG2 in a unique in vitro model of CRC progression, to assess by which mechanisms it acts a tumour suppressor, and how TG2 expression is regulated.The in vitro CRC model used consisted of two cell lines: SW480 (derived by the primary tumour site of a CRC patient and expressing very high TG2), and SW620 (derived by a lymph node metastasis of the same patient and expressing very little TG2).Silencing of TG2 in SW480 directly promoted cell invasion on Transwell system, whereas transfecting TG2 in SW620 prevented it. Compared to SW620, in SW480 TG2 is found more SUMOylated at the leading edges of cells, and TG2 levels are positively correlated with ?-catenin levels, suggesting a role for TG2 in maintaining cell-cell junctions and regulating motility. Silencing TG2 in SW480 and transfecting TG2 in SW620 show that TG2 levels are positively correlated with expression of HLA?I; this effect may be linked to tumour immune evasion. To understand how TG2 expression is regulated, in silico and experimental analysis were performed which identified miR-19a as a regulator. Transfection of miR-19a in SW480 directly downregulated TG2 resulting in cell invasion.Given that exogenous administration of TG2 would not represent a viable option due to its systemic expression and pleiotropic functions, these observations provide a rationale for sequestering miR-19a in primary CRC tumour in order to prevent downregulation of TG2 and thus metastasis.<br/

Dysregulation of anti-viral function of CD8+T cells in the COPD lung: role of the PD1/PDL1 axis

Rationale: COPD patients are susceptible to respiratory viral infections which cause exacerbations. Mechanisms underlying susceptibility are not understood. Effectors of the adaptive immune response; CD8+ T cells which clear viral infections, are present in increased numbers in lungs of COPD patients but fail to protect against infection and may contribute to the immunopathology of the disease. Objectives: CD8+ function and signalling through the Programmed Cell Death (PD-1) exhaustion pathway was investigated as a potential key mechanism of viral exacerbation of the COPD lung. Methods: Tissue from control or COPD patients undergoing lung resection was infected with live influenza virus ex vivo. Viral infection and expression of lung cell markers was analysed using flow cytometry. Measurements and Main Results: The proportion of lung CD8+ T cells expressing PD-1 was greater in COPD(mean=16.2%) than controls(4.4%, p=0.029). Only epithelial cells and macrophages were infected with influenza and there was no difference in the proportion of infected cells between controls and COPD. Infection upregulated T cell PD-1 expression in control and COPD samples. Concurrently, influenza significantly upregulated the marker of cytotoxic degranulation (CD107a) on CD8+ T cells(p=0.03) from controls, but not from COPD patients. Virus-induced expression of the ligand PD-L1 was decreased on COPD macrophages(p=0.04) with a corresponding increase in IFN? release from infected COPD explants compared to controls(p=0.04). Conclusions: This study has established a signal of cytotoxic immune dysfunction and aberrant immune regulation in the COPD lung that may explain both the susceptibility to viral infection and the excessive, inflammation associated with exacerbations

IFN-γ influences epithelial anti-viral responses via histone methylation of the RIG-I promoter

The asthmatic lung is prone to respiratory viral infections that exacerbate the symptoms of the underlying disease. Recent work has suggested that a deficient Th1 response in early life may lead to these aberrant anti-viral responses. We investigated whether the inflammatory environment of the airway epithelium could modulate anti-viral gene expression via epigenetic mechanisms, in order to study the development of a long-term dysregulation of innate responses, which are a hallmark of asthma.We primed AALEB, a human bronchial epithelial cell line, with IFNγ and IL13 and subsequently infected cells with Respiratory Syncytial Virus (RSV) and innate anti-viral genes expression and their epigenetic markers were analysed.Priming epithelial cells with IFNγ reduced RSV viral load. Microarray analysis identified that IFNγ-priming enhanced RIG-I mRNA expression and this expression correlated with epigenetic changes at the RIG-I promoter that influenced its transcription. Using chromatin immunoprecipitation, we observed a reduction of trimethylated Histone3 Lysine9 (H3K9me3) at the RIG-I promoter. Addition of inhibitor BIX-01294 to this model indicated an involvement of lysine methyltransferase G9a in RIG-I epigenetic regulation.These data suggest that prior exposure to IFNγ may leave an epigenetic mark upon the chromatin that enhances airway cells’ ability to better resist infection possibly via epigenetic upregulation of RIG-I. These observations provide further evidence for a crucial role of IFNγ in the development of mature anti-viral responses within a model of respiratory infection. Further clinical validation is required to determine if this effect in early life leads to changes in anti-viral responses associated with asthma

Dynamics of IFN-β responses during respiratory viral infection: insights for therapeutic strategies

RATIONALE: Viral infections are major drivers of exacerbations and clinical burden in patients with asthma and COPD. IFN-β is a key component of the innate immune response to viral infection. To date studies of inhaled IFN-β treatment have not demonstrated a significant effect on asthma exacerbations. OBJECTIVES: The dynamics of exogenous IFN-β activity were investigated to inform on future clinical indications for this potential anti-viral therapy. METHODS: Monocyte-derived macrophages (MDMs), alveolar macrophages (AMs) and primary bronchial epithelial cells (PBECs) were isolated from healthy controls and COPD patients and infected with influenza virus either prior to or after IFN-β stimulation. Infection levels were measured by % nucleoprotein 1 positive (NP1+) cells using flow cytometry. Viral RNA shedding and interferon stimulated gene expression were measured by qPCR. Production of inflammatory cytokines was measured using MSD. MEASUREMENTS AND MAIN RESULTS: Adding IFN-β to MDMs, AMs and PBECs prior to, but not after, infection reduced %NP1+ cells by 85%, 56% and 66%, respectively (p&lt;0.05). Inhibition of infection lasted for 24h following removal of IFN-β and was maintained albeit reduced up to 1 week in MDMs and 72h in PBECs; this was similar between health and COPD. IFN-β did not induce inflammatory cytokine production by MDMs or PBECs but reduced influenza-induced IL-1β production by PBECs. CONCLUSIONS: In vitro modelling of IFN-β dynamics highlights the potential for intermittent prophylactic doses of exogenous IFN-β to modulate viral infection. This provides important insights to aid the future design of clinical trials of IFN-β in asthma and COPD

Pulmonary EV miRNA profiles identify disease and distinct inflammatory endotypes in COPD

IntroductionChronic obstructive pulmonary disease (COPD) is a heterogeneous condition without effective disease modifying therapies. Identification of novel inflammatory endotype markers such as extracellular vesicles (EVs), which are important intercellular messengers carrying microRNA (miRNA), may enable earlier diagnosis and disease stratification for a targeted treatment approach. Our aim was to identify differentially expressed EV miRNA in the lungs of COPD patients compared with healthy ex-smokers and determine whether they can help define inflammatory COPD endotypes.MethodsEV miRNA were isolated and sequenced from ex-smoking COPD patients and healthy ex-smoker bronchoalveolar lavage fluid. Results were validated with RT-qPCR and compared to differential inflammatory cell counts.ResultsExpression analysis identified five upregulated miRNA in COPD (miR-223-3p, miR-2110, miR-182-5p, miR-200b-5p and miR-625-3p) and three downregulated miRNA (miR-138-5p, miR-338-3p and miR-204-5p), all with a log2 fold change of &gt;1/-1, FDR &lt; 0.05. These miRNAs correlated with disease defining characteristics such as FEF 25-75% (a small airways disease measure) and DLCO% (a surrogate measure of emphysema). Receiver operator curve analysis demonstrated miR-2110, miR-223-3p, and miR-182-5p showed excellent combinatory predictive ability (AUC 0.91, p &lt; 0.0001) in differentiating between health and mild COPD. Furthermore, miR-223-3p and miR-338-3p correlated with airway eosinophilia and were able to distinguish "pure eosinophilic" COPD from other airway inflammatory subtypes (AUC 0.94 and 0.85, respectively).DiscussionThis is the first study to identify differentially expressed miRNA in COPD bronchoalveolar lavage fluid EVs. These findings suggest specific lung derived EV miRNA are a strong predictor of disease presence even in mild COPD. Furthermore, specific miRNA correlated with inflammatory cell numbers in COPD, and may have a role in defining inflammatory endotypes for future treatment stratification

Exercise training induces a shift in extracellular redox status with alterations in the pulmonary and systemic redox landscape in asthma

Redox dysregulation and oxidative stress have been implicated in asthma pathogenesis. Exercise interventions improve symptoms and reduce inflammation in asthma patients, but the underlying mechanisms remain unclear. We hypothesized that a personalised exercise intervention would improve asthma control by reducing lung inflammation through modulation of local and systemic reactive species interactions, thereby increasing antioxidant capacity. We combined deep redox metabolomic profiling with clinical assessment in an exploratory cohort of six female patients with symptomatic asthma and studied their responses to a metabolically targeted exercise intervention over 12 weeks. Plasma antioxidant capacity and circulating nitrite levels increased following the intervention (p = 0.028) and lowered the ratio of reduced to oxidised glutathione (p = 0.029); this was accompanied by improvements in physical fitness (p = 0.046), symptoms scores (p = 0.020), quality of life (p = 0.046), lung function (p = 0.028), airway hyperreactivity (p = 0.043), and eosinophilic inflammation (p = 0.007). Increased physical fitness correlated with improved plasma antioxidant capacity (p = 0.019), peak oxygen uptake and nitrite changes (p = 0.005), the latter also associated with reductions in peripheral blood eosinophil counts (p = 0.038). Thus, increases in “redox resilience” may underpin the clinical benefits of exercise in asthma. An improved understanding of exercise-induced alterations in redox regulation offers opportunities for greater treatment personalisation and identification of new treatment targets.</p

Dysregulation of COVID-19 related gene expression in the COPD lung

Background: chronic obstructive pulmonary disease (COPD) patients are at increased risk of poor outcome from COVID-19. Early data suggest increased expression of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) receptor angiotensin converting enzyme 2 (ACE2), but relationships to disease phenotype and downstream regulators of inflammation in the Renin-Angiotensin system (RAS) are unknown. To determine the relationship between RAS gene expression relevant to SARS-CoV-2 infection in the lung and relationship to disease characteristics in COPD.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 biopsies (0.23, p=0.050), and correlated with worse lung function (r=-0.28, p=0.0090). Angiotensin converting enzyme 2 (ACE2) was further increased in frequent exacerbators compared to infrequent exacerbators (0.51, p=0.00045). Increased ACE2 expression also associated with use of ACE inhibitors (ACEi) (0.50, p=0.0034) and having cardiovascular disease (0.23, p=0.048) or hypertension (0.34, p=0.0089) and inhaled corticosteroid use in COPD subjects in biopsies (0.33, p=0.049). Angiotensin II receptor type (AGTR)1 and 2 expression was decreased in COPD 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).Conclusion: this analysis reveals the potential mechanisms driving susceptibility to SARS-CoV-2 infection and inflammation associated with severe COVID-19 seen in COPD. Extension of this approach to other viruses may lead to opportunities for therapeutic development to improve outcomes in the pandemic and beyond

Data_Sheet_1_Pulmonary EV miRNA profiles identify disease and distinct inflammatory endotypes in COPD.PDF

IntroductionChronic obstructive pulmonary disease (COPD) is a heterogeneous condition without effective disease modifying therapies. Identification of novel inflammatory endotype markers such as extracellular vesicles (EVs), which are important intercellular messengers carrying microRNA (miRNA), may enable earlier diagnosis and disease stratification for a targeted treatment approach. Our aim was to identify differentially expressed EV miRNA in the lungs of COPD patients compared with healthy ex-smokers and determine whether they can help define inflammatory COPD endotypes.MethodsEV miRNA were isolated and sequenced from ex-smoking COPD patients and healthy ex-smoker bronchoalveolar lavage fluid. Results were validated with RT-qPCR and compared to differential inflammatory cell counts.ResultsExpression analysis identified five upregulated miRNA in COPD (miR-223-3p, miR-2110, miR-182-5p, miR-200b-5p and miR-625-3p) and three downregulated miRNA (miR-138-5p, miR-338-3p and miR-204-5p), all with a log2 fold change of >1/−1, FDR DiscussionThis is the first study to identify differentially expressed miRNA in COPD bronchoalveolar lavage fluid EVs. These findings suggest specific lung derived EV miRNA are a strong predictor of disease presence even in mild COPD. Furthermore, specific miRNA correlated with inflammatory cell numbers in COPD, and may have a role in defining inflammatory endotypes for future treatment stratification.</p