Cytokine biomarkers of exacerbations in sputum from Chronic Obstructive Pulmonary Disease patients: a prospective cohort study.

BACKGROUND: We determined the relationships between cytokine expression in sputum and clinical data to characterise and understand Chronic Obstructive Pulmonary Disease (COPD) exacerbations in COPD patients. METHODS: We measured 30 cytokines in 936 sputum samples, collected at stable state (ST) and exacerbation (EX) visits from 99 participants in the Acute Exacerbation and Respiratory InfectionS in COPD (AERIS) study (NCT01360398, www.clinicaltrials.gov). We determined their longitudinal expression and examined differential expression based on disease status or exacerbation type. RESULTS: Of the cytokines, 17 were suitable for analysis. As for disease states, in EX sputum samples, IL-17A, TNF-α, IL-1ß, and IL-10 were significantly increased compared to ST sputum samples, but a logistic mixed model could not predict disease state. As for exacerbation types, bacteria-associated exacerbations showed higher expression of IL-17A, TNF-α, IL-1ß, and IL-1α. IL-1α, IL-1ß, and TNF-α were identified as suitable biomarkers for bacteria-associated exacerbation. Bacteria-associated exacerbations also formed a cluster separate from other exacerbation types in principal component analysis. CONCLUSIONS: Measurement of cytokines in sputum from COPD patients could help identify bacteria-associated exacerbations based on increased concentrations of IL-1α, IL-1ß, or TNF-α. This finding may provide a point-of-care assessment to distinguish a bacterial exacerbation of COPD from other exacerbation types.

Antiviral Responses of Tissue-resident CD49a+ Lung Natural Killer Cells Are Dysregulated in Chronic Obstructive Pulmonary Disease.

Rationale: Tissue-resident natural killer (trNK) cells have been identified in numerous organs, but little is known about their functional contribution to respiratory immunity, in particular during chronic lung diseases such as chronic obstructive pulmonary disease (COPD). Objectives: To investigate the phenotype and antiviral responses of trNK cells in murine cigarette smoke-induced experimental COPD and in human lung parenchyma from COPD donors. Methods: Mice were exposed to cigarette smoke for 12 weeks to induce COPD-like lung disease. Lung trNK cell phenotypes and function were analyzed by flow cytometry in both murine and human disease with and without challenge with influenza A virus. Measurements and Main Results: In the mouse lung, CD49a+CD49b+EOMES+ and CD49a+CD49b-EOMESlo NK cell populations had a distinct phenotype compared with CD49a- circulating NK cells. CD49a+ NK cells were more extensively altered earlier in disease onset than circulating NK cells, and increased proportions of CD49a+ NK cells correlated with worsening disease in both murine and human COPD. Furthermore, the presence of lung disease delayed both circulating and trNK cell functional responses to influenza infection. CD49a+ NK cells markedly increased their NKG2D, CD103, and CD69 expression in experimental COPD after influenza infection, and human CD49a+ NK cells were hyperactive to ex vivo influenza infection in COPD donors. Conclusions: Collectively, these results demonstrate that trNK cell function is altered in cigarette smoke-induced disease and suggests that smoke exposure may aberrantly prime trNK cell responsiveness to viral infection. This may contribute to excess inflammation during viral exacerbations of COPD.

Towards an artificial human lung: modelling organ-like complexity to aid mechanistic understanding.

Respiratory diseases account for over 5 million deaths yearly and are a huge burden to healthcare systems worldwide. Murine models have been of paramount importance to decode human lung biology in vivo, but their genetic, anatomical, physiological and immunological differences with humans significantly hamper successful translation of research into clinical practice. Thus, to clearly understand human lung physiology, development, homeostasis and mechanistic dysregulation that may lead to disease, it is essential to develop models that accurately recreate the extraordinary complexity of the human pulmonary architecture and biology. Recent advances in micro-engineering technology and tissue engineering have allowed the development of more sophisticated models intending to bridge the gap between the native lung and its replicates in vitro Alongside advanced culture techniques, remarkable technological growth in downstream analyses has significantly increased the predictive power of human biology-based in vitro models by allowing capture and quantification of complex signals. Refined integrated multi-omics readouts could lead to an acceleration of the translational pipeline from in vitro experimental settings to drug development and clinical testing in the future. This review highlights the range and complexity of state-of-the-art lung models for different areas of the respiratory system, from nasal to large airways, small airways and alveoli, with consideration of various aspects of disease states and their potential applications, including pre-clinical drug testing. We explore how development of optimised physiologically relevant in vitro human lung models could accelerate the identification of novel therapeutics with increased potential to translate successfully from the bench to the patient's bedside.

Nontypeable Haemophilus influenzae infection of pulmonary macrophages drives neutrophilic inflammation in severe asthma.

BACKGROUND: Nontypeable Haemophilus influenzae (NTHi) is a respiratory tract pathobiont that chronically colonizes the airways of asthma patients and is associated with severe, neutrophilic disease phenotypes. The mechanism of NTHi airway persistence is not well understood, but accumulating evidence suggests NTHi can persist within host airway immune cells such as macrophages. We hypothesized that NTHi infection of pulmonary macrophages drives neutrophilic inflammation in severe asthma. METHODS: Bronchoalveolar lavage (BAL) samples from 25 severe asthma patients were assessed by fluorescence in situ hybridisation to quantify NTHi presence. Weighted gene correlation network analysis (WGCNA) was performed on RNASeq data from NTHi-infected monocyte-derived macrophages to identify transcriptomic networks associated with NTHi infection. RESULTS: NTHi was detected in 56% of BAL samples (NTHi+) and was associated with longer asthma duration (34 vs 22.5 years, p = .0436) and higher sputum neutrophil proportion (67% vs 25%, p = .0462). WGCNA identified a transcriptomic network of immune-related macrophage genes significantly associated with NTHi infection, including upregulation of T17 inflammatory mediators and neutrophil chemoattractants IL1B, IL8, IL23 and CCL20 (all p < .05). Macrophage network genes SGPP2 (p = .0221), IL1B (p = .0014) and GBP1 (p = .0477) were more highly expressed in NTHi+ BAL and moderately correlated with asthma duration (IL1B; rho = 0.41, p = .041) and lower prebronchodilator FEV1/FVC% (GBP1; rho = -0.43, p = .046 and IL1B; rho = -0.42, p = .055). CONCLUSIONS: NTHi persistence with pulmonary macrophages may contribute to chronic airway inflammation and T17 responses in severe asthma, which can lead to decreased lung function and reduced steroid responsiveness. Identifying therapeutic strategies to reduce the burden of NTHi in asthma could improve patient outcomes.

Inflammatory Endotype-associated Airway Microbiome in Chronic Obstructive Pulmonary Disease Clinical Stability and Exacerbations: A Multicohort Longitudinal Analysis.

Rationale: Understanding the role of the airway microbiome in chronic obstructive pulmonary disease (COPD) inflammatory endotypes may help to develop microbiome-based diagnostic and therapeutic approaches. Objectives: To understand the association of the airway microbiome with neutrophilic and eosinophilic COPD at stability and during exacerbations. Methods: An integrative analysis was performed on 1,706 sputum samples collected longitudinally from 510 patients with COPD recruited at four UK sites of the BEAT-COPD (Biomarkers to Target Antibiotic and Systemic COPD), COPDMAP (Chronic Obstructive Pulmonary Disease Medical Research Council/Association of the British Pharmaceutical Industry), and AERIS (Acute Exacerbation and Respiratory Infections in COPD) cohorts. The microbiome was analyzed using COPDMAP and AERIS as a discovery data set and BEAT-COPD as a validation data set. Measurements and Main Results: The airway microbiome in neutrophilic COPD was heterogeneous, with two primary community types differentiated by the predominance of Haemophilus. The Haemophilus-predominant subgroup had elevated sputum IL-1ß and TNFα (tumor necrosis factor α) and was relatively stable over time. The other neutrophilic subgroup with a balanced microbiome profile had elevated sputum and serum IL-17A and was temporally dynamic. Patients in this state at stability were susceptible to the greatest microbiome shifts during exacerbations. This subgroup can temporally switch to both neutrophilic Haemophilus-predominant and eosinophilic states that were otherwise mutually exclusive. Time-series analysis on the microbiome showed that the temporal trajectories of Campylobacter and Granulicatella were indicative of intrapatient switches from neutrophilic to eosinophilic inflammation, in track with patient sputum eosinophilia over time. Network analysis revealed distinct host-microbiome interaction patterns among neutrophilic Haemophilus-predominant, neutrophilic balanced microbiome, and eosinophilic subgroups. Conclusions: The airway microbiome can stratify neutrophilic COPD into subgroups that justify different therapies. Neutrophilic and eosinophilic COPD are interchangeable in some patients. Monitoring temporal variability of the airway microbiome may track patient inflammatory status over time.

Biomarkers to guide the use of antibiotics for acute exacerbations of COPD (AECOPD): a systematic review and meta-analysis.

BACKGROUND: Antibiotics are frequently prescribed for acute exacerbations of COPD (AECOPD) even though most do not have a bacterial aetiology. Biomarkers may help clinicians target antibiotic use by identifying AECOPD caused by bacterial pathogens. We aimed to summarise current evidence on the diagnostic accuracy of biomarkers for detecting bacterial versus non-bacterial AECOPD. METHODS: We searched Embase and Medline using a search strategy including terms for COPD, biomarkers and bacterial infection. Data regarding diagnostic accuracy for each biomarker in predicting bacterial cause of exacerbation were extracted and summarised. We used to QUADAS-2 tool to assess risk of bias. RESULTS: Of 509 papers identified, 39 papers evaluating 61 biomarkers were eligible for inclusion. Moderate quality evidence was found for associations between serum C-reactive protein (CRP), serum procalcitonin (PCT), sputum interleukin (IL)-8 and sputum tumour necrosis factor alpha (TNF-α), and the presence of bacterial pathogens in the sputum of patients with AECOPD. Having bacterial pathogens was associated with a mean difference (higher) CRP and PCT of 29.44 mg/L and 0.76 ng/mL respectively. There was inconsistent or weak evidence for associations between bacterial AECOPD and higher levels of sputum IL-1ß, IL-6, myeloperoxidase (MPO) and neutrophil elastase (NE). We did not find any consistent evidence of diagnostic value for other biomarkers. CONCLUSIONS: There is moderate evidence from heterogeneous studies that serum CRP and PCT are of value in differentiating bacterial from non-bacterial AECOPD, and little evidence for other biomarkers. Further high-quality research on the role of biomarkers in identifying bacterial exacerbations is needed.

Sputum sample positivity for Haemophilus influenzae or Moraxella catarrhalis in acute exacerbations of chronic obstructive pulmonary disease: evaluation of association with positivity at earlier stable disease timepoints.

BACKGROUND: Infection with Haemophilus influenzae (Hi) or Moraxella catarrhalis (Mcat) is a risk factor for exacerbation in chronic obstructive pulmonary disease (COPD). The ability to predict Hi- or Mcat-associated exacerbations may be useful for interventions developed to reduce exacerbation frequency. METHODS: In a COPD observational study, sputum samples were collected at monthly stable-state visits and at exacerbation during two years of follow-up. Bacterial species (Hi, Mcat) were identified by culture and quantitative PCR assay. Post-hoc analyses were conducted to assess: (1) first Hi- or Mcat-positive exacerbations given presence or absence of Hi or Mcat at the screening visit (stable-state timepoint); (2) first Hi- or Mcat-positive exacerbations given presence or absence of Hi or Mcat at stable timepoints within previous 90 days; (3) second Hi- or Mcat-positive exacerbations given presence or absence of Hi or Mcat at stable timepoints within previous 90 days. Percentages and risk ratios (RRs) with 95% confidence intervals were calculated. RESULTS: PCR results for analyses 1, 2 and 3 (samples from 84, 88 and 83 subjects, respectively) showed that the risk of an Hi- or Mcat-positive exacerbation is significantly higher if sputum sample was Hi- or Mcat-positive than if Hi- or Mcat-negative at previous stable timepoints (apart from Mcat in analysis 3); RRs ranged from 2.1 to 3.2 for Hi and 1.9 to 2.6 for Mcat.For all analyses, the percentage of Hi- or Mcat-positive exacerbations given previous Hi- or Mcat-positive stable timepoints was higher than the percentage of Hi- or Mcat-positive exacerbations if Hi- or Mcat-negative at previous stable timepoints. Percentage of Hi- or Mcat-positive exacerbations given previous Hi- or Mcat-negative stable timepoints was 26.3%-37.0% for Hi and 17.6%-19.7% for Mcat. CONCLUSIONS: Presence of Hi or Mcat at a stable timepoint was associated with a higher risk of a subsequent Hi- or Mcat-associated exacerbation compared with earlier absence. However, a large percentage of Hi- or Mcat-associated exacerbations was not associated with Hi/Mcat detection at an earlier timepoint. This suggests that administration of an intervention to reduce these exacerbations should be independent of bacterial presence at baseline. Trial Registration https://clinicaltrials.gov/ ; NCT01360398, registered May 25, 2011.

Seroprevalence of Bordetella pertussis Infection in Patients With Chronic Obstructive Pulmonary Disease in England: Analysis of the AERIS Cohort.

Pertussis is underdiagnosed and underreported in adults and patients with underlying conditions. Patients with chronic obstructive pulmonary disease (COPD) may be at increased risk of severe pertussis. Understanding the true prevalence of pertussis infections in such patients is important. We therefore evaluated the seroprevalence of anti-pertussis toxin (PT) antibodies in a cohort of 40-85-year-old patients diagnosed with moderate, severe or very severe COPD enrolled (between June 2011 and June 2012) in the prospective, observational "Acute Exacerbation and Respiratory InfectionS in COPD" (AERIS; NCT01360398) study, conducted in England. Serum anti-PT antibodies were measured in 104 patients using an enzyme-linked immunosorbent assay on samples collected 12 months (M12) and 24 months (M24) after enrollment. Overall, 14/104 (13.5%) patients had anti-PT concentrations ≥50 IU/mL at M12 or M24, indicative of exposure to Bordetella pertussis during the preceding 2-3 years. Of these, 6/104 (5.8%) had anti-PT ≥70 IU/mL, of whom 3/104 (2.9%) had anti-PT ≥120 IU/mL, indicative of exposure within 12 and 6 months, respectively. These results show a high circulation of B. pertussis in 40-85-year-old patients with moderate, severe or very severe COPD in England between 2012 and 2014, and call for enhanced immunization to prevent pertussis infections in such patients.

Human Lung Fibroblasts Present Bacterial Antigens to Autologous Lung Th Cells.

Lung fibroblasts are key structural cells that reside in the submucosa where they are in contact with large numbers of CD4+ Th cells. During severe viral infection and chronic inflammation, the submucosa is susceptible to bacterial invasion by lung microbiota such as nontypeable Haemophilus influenzae (NTHi). Given their proximity in tissue, we hypothesized that human lung fibroblasts play an important role in modulating Th cell responses to NTHi. We demonstrate that fibroblasts express the critical CD4+ T cell Ag-presentation molecule HLA-DR within the human lung, and that this expression can be recapitulated in vitro in response to IFN-γ. Furthermore, we observed that cultured lung fibroblasts could internalize live NTHi. Although unable to express CD80 and CD86 in response to stimulation, fibroblasts expressed the costimulatory molecules 4-1BBL, OX-40L, and CD70, all of which are related to memory T cell activation and maintenance. CD4+ T cells isolated from the lung were predominantly (mean 97.5%) CD45RO+ memory cells. Finally, cultured fibroblasts activated IFN-γ and IL-17A cytokine production by autologous, NTHi-specific lung CD4+ T cells, and cytokine production was inhibited by a HLA-DR blocking Ab. These results indicate a novel role for human lung fibroblasts in contributing to responses against bacterial infection through activation of bacteria-specific CD4+ T cells.

IL-12 and IL-7 synergize to control mucosal-associated invariant T-cell cytotoxic responses to bacterial infection.

BACKGROUND: Bacterial respiratory tract infections and exacerbations of chronic lung diseases are commonly caused by nontypeable Haemophilus influenzae (NTHi). Cell-mediated cytotoxicity might be key to controlling infection, but the responses of NTHi-specific T-cell populations are not well understood. Mucosal-associated invariant T (MAIT) cells are a recently discovered, innate-like subset of T cells with cytotoxic function, the role of which in lung immunity is unclear. OBJECTIVE: The aim of this study was to determine the mechanisms behind conventional T-cell and MAIT cell cytotoxic responses to NTHi. METHODS: Human ex vivo lung explants were infected with a clinical strain of NTHi. Monocyte-derived macrophages were also infected with NTHi in vitro and cocultured with autologous T cells. Cytotoxic responses of T-cell subsets were measured by using flow cytometry. RESULTS: We found significant upregulation of the cytotoxic markers CD107a and granzyme B in lung CD4+, CD8+, and MAIT cell populations. We show that MAIT cell cytotoxic responses were upregulated by a combination of both time-dependent antigen presentation and a novel mechanism through which IL-12 and IL-7 synergistically control granzyme B through upregulation of the IL-12 receptor. CONCLUSIONS: Overall, our data provide evidence for a cytotoxic role of MAIT cells in the lung and highlight important differences in the control of adaptive and innate-like T-cell responses. Understanding these mechanisms might lead to new therapeutic opportunities to modulate the antibacterial response and improve clinical outcome.

Longitudinal profiling of the lung microbiome in the AERIS study demonstrates repeatability of bacterial and eosinophilic COPD exacerbations.

BACKGROUND: Alterations in the composition of the lung microbiome associated with adverse clinical outcomes, known as dysbiosis, have been implicated with disease severity and exacerbations in COPD. OBJECTIVE: To characterise longitudinal changes in the lung microbiome in the AERIS study (Acute Exacerbation and Respiratory InfectionS in COPD) and their relationship with associated COPD outcomes. METHODS: We surveyed 584 sputum samples from 101 patients with COPD to analyse the lung microbiome at both stable and exacerbation time points over 1 year using high-throughput sequencing of the 16S ribosomal RNA gene. We incorporated additional lung microbiology, blood markers and in-depth clinical assessments to classify COPD phenotypes. RESULTS: The stability of the lung microbiome over time was more likely to be decreased in exacerbations and within individuals with higher exacerbation frequencies. Analysis of exacerbation phenotypes using a Markov chain model revealed that bacterial and eosinophilic exacerbations were more likely to be repeated in subsequent exacerbations within a subject, whereas viral exacerbations were not more likely to be repeated. We also confirmed the association of bacterial genera, including Haemophilus and Moraxella, with disease severity, exacerbation events and bronchiectasis. CONCLUSIONS: Subtypes of COPD have distinct bacterial compositions and stabilities over time. Some exacerbation subtypes have non-random probabilities of repeating those subtypes in the future. This study provides insights pertaining to the identification of bacterial targets in the lung and biomarkers to classify COPD subtypes and to determine appropriate treatments for the patient. TRIAL REGISTRATION NUMBER: Results, NCT01360398.

Development of flow cytometric opsonophagocytosis and antibody-mediated complement deposition assays for non-typeable Haemophilus influenzae.

BACKGROUND: Haemophilus influenzae is found in the nasopharynx of 80% of the human population. While colonisation with non-typeable Haemophilus influenzae (NTHi) is usually asymptomatic, it is capable of causing acute and chronic otitis media (OM) in infants, invasive disease in susceptible groups and is the leading cause of exacerbations of patients with chronic obstructive pulmonary disease (COPD). Current methods for assessing functional antibody immunity to NTHi are limited and labour intensive. Flow cytometric assays could provide an attractive alternative to evaluate immune responses to candidate vaccines in clinical trials. RESULTS: We have developed a duplexed flow-cytometric uptake and oxidative burst opsonophagocytosis assay (fOPA). We have also developed a duplexed antibody-mediated complement C3b/iC3b and C5b-9 deposition assay (CDA). Antibody-mediated C3b/iC3b deposition correlated with opsonophagocytic uptake (r = 0.65) and with opsonophagocytic oxidative burst (r = 0.69). Both fOPA and CDA were reproducible, with the majority of samples giving a coefficient of variation (CV) of < 20% and overall assay CVs of 14% and 16% respectively. CONCLUSIONS: The high-throughput flow cytometric assays developed here were successfully optimised for use with NTHi. Assays proved to be sensitive and highly reproducible for the measurement of bacterial uptake and oxidative burst opsonophagocytosis and antibody-mediated deposition of C3b/iC3b and C5b-9. These assays are useful tools for use in large scale epidemiological studies and to assist in the assessment of functional antibody induced by NTHi candidate vaccines.

Impact of radiologically stratified exacerbations: insights into pneumonia aetiology in COPD.

BACKGROUND: COPD patients have increased risk of developing pneumonia, which is associated with poor outcomes. It can be symptomatically indistinguishable from exacerbations, making diagnosis challenging. Studies of pneumonia in COPD have focused on hospitalised patients and are not representative of the ambulant COPD population. Therefore, we sought to determine the incidence and aetiology of acute exacerbation events with evidence of pneumonic radiographic infiltrates in an outpatient COPD cohort. METHODS: One hundred twenty-seven patients with moderate to very severe COPD aged 42-85 years underwent blood and sputum sampling over one year, at monthly stable visits and within 72 h of exacerbation symptom onset. 343 exacerbations with chest radiographs were included. RESULTS: 20.1% of exacerbations had pneumonic infiltrates. Presence of infiltrate was highly seasonal (Winter vs summer OR 3.056, p = 0.027). In paired analyses these exacerbation events had greater increases in systemic inflammation. Bacterial detection rate was higher in the pneumonic group, with Haemophilus influenzae the most common bacteria in both radiological groups. Viral detection and sputum microbiota did not differ with chest radiograph appearance. CONCLUSIONS: In an outpatient COPD cohort, pneumonic infiltrates at exacerbation were common, and associated with more intense inflammation. Bacterial pathogen detection and lung microbiota were not distinct, suggesting that exacerbations and pneumonia in COPD share common infectious triggers and represent a continuum of severity rather than distinct aetiological events. TRIAL REGISTRATION: Trial registration Number: NCT01360398 .

Relationship of CT-quantified emphysema, small airways disease and bronchial wall dimensions with physiological, inflammatory and infective measures in COPD.

BACKGROUND: COPD is a complex, heterogeneous disease characterised by progressive development of airflow limitation. Spirometry provides little information about key aspects of pathology and is poorly related to clinical outcome, so other tools are required to investigate the disease. We sought to explore the relationships between quantitative CT analysis with functional, inflammatory and infective assessments of disease to identify the utility of imaging to stratify disease to better predict outcomes and disease response. METHODS: Patients from the AERIS study with moderate-very severe COPD underwent HRCT, with image analysis determining the quantity of emphysema (%LAA<- 950), small airways disease (E/I MLD) and bronchial wall thickening (Pi10). At enrolment subjects underwent lung function testing, six-minute walk testing (6MWT), blood sampling for inflammatory markers and sputum sampling for white cell differential and microbiological culture and PCR. RESULTS: 122 subjects were included in this analysis. Emphysema and small airways disease had independent associations with airflow obstruction (ß = - 0.34, p < 0.001 and ß = - 0.56, p < 0.001). %LAA<- 950 had independent associations with gas transfer (ß = - 0.37, p < 0.001) and E/I MLD with RV/TLC (ß = 0.30, p =0.003). The distance walked during the 6MWT was not associated with CT parameters, but exertional desaturation was independently associated with emphysema (ß = 0.73, p < 0.001). Pi10 did not show any independent associations with lung function or functional parameters. No CT parameters had any associations with sputum inflammatory cells. Greater emphysema was associated with lower levels of systemic inflammation (CRP ß = - 0.34, p < 0.001 and fibrinogen ß = - 0.28, p =0.003). There was no significant difference in any of the CT parameters between subjects where potentially pathogenic bacteria were detected in sputum and those where it was not. CONCLUSIONS: This study provides further validation for the use of quantitative CT measures of emphysema and small airways disease in COPD as they showed strong associations with pulmonary physiology and functional status. In contrast to this quantitative CT measures showed few convincing associations with biological measures of disease, suggesting it is not an effective tool at measuring disease activity.

IFN-γ Influences Epithelial Antiviral 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 T-helper cell type 1 response in early life may lead to these aberrant antiviral responses. To study the development of long-term dysregulation of innate responses, which is a hallmark of asthma, we investigated whether the inflammatory environment of the airway epithelium can modulate antiviral gene expression via epigenetic mechanisms. We primed AALEB cells, a human bronchial epithelial cell line, with IFN-γ and IL-13, and subsequently infected the cells with respiratory syncytial virus (RSV). We then analyzed the expression of innate antiviral genes and their epigenetic markers. Priming epithelial cells with IFN-γ reduced the RSV viral load. Microarray analysis identified that IFN-γ priming enhanced retinoic acid-inducible gene (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 histone 3 lysine 9 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 IFN-γ may leave an epigenetic mark on the chromatin that enhances airway cells' ability to resist infection, possibly via epigenetic upregulation of RIG-I. These observations provide further evidence for a crucial role of IFN-γ in the development of mature antiviral responses within a model of respiratory infection. Further clinical validation is required to determine whether this effect in early life leads to changes in antiviral responses associated with asthma.

A prospective, observational cohort study of the seasonal dynamics of airway pathogens in the aetiology of exacerbations in COPD.

BACKGROUND: The aetiology of acute exacerbations of COPD (AECOPD) is incompletely understood. Understanding the relationship between chronic bacterial airway infection and viral exposure may explain the incidence and seasonality of these events. METHODS: In this prospective, observational cohort study (NCT01360398), patients with COPD aged 40-85 years underwent sputum sampling monthly and at exacerbation for detection of bacteria and viruses. Results are presented for subjects in the full cohort, followed for 1 year. Interactions between exacerbation occurrence and pathogens were investigated by generalised estimating equation and stratified conditional logistic regression analyses. FINDINGS: The mean exacerbation rate per patient-year was 3.04 (95% CI 2.63 to 3.50). At AECOPD, the most common bacterial species were non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis, and the most common virus was rhinovirus. Logistic regression analyses (culture bacterial detection) showed significant OR for AECOPD occurrence when M. catarrhalis was detected regardless of season (5.09 (95% CI 2.76 to 9.41)). When NTHi was detected, the increased risk of exacerbation was greater in high season (October-March, OR 3.04 (1.80 to 5.13)) than low season (OR 1.22 (0.68 to 2.22)). Bacterial and viral coinfection was more frequent at exacerbation (24.9%) than stable state (8.6%). A significant interaction was detected between NTHi and rhinovirus presence and AECOPD risk (OR 5.18 (1.92 to 13.99); p=0.031). CONCLUSIONS: AECOPD aetiology varies with season. Rises in incidence in winter may be driven by increased pathogen presence as well as an interaction between NTHi airway infection and effects of viral infection. TRIAL REGISTRATION NUMBER: Results, NCT01360398.

Impact and associations of eosinophilic inflammation in COPD: analysis of the AERIS cohort.

Eosinophilic inflammation in chronic obstructive pulmonary disease (COPD) predicts response to treatment, especially corticosteroids. We studied the nature of eosinophilic inflammation in COPD prospectively to examine the stability of this phenotype and its dynamics across exacerbations, and its associations with clinical phenotype, exacerbations and infection.127 patients aged 40-85 years with moderate to very severe COPD underwent repeated blood and sputum sampling at stable visits and within 72 h of exacerbation for 1 year.Blood eosinophils ≥2% was prevalent at baseline, and predicted both predominantly raised stable-state eosinophils across the year (area under the curve 0.841, 95% CI 0.755-0.928) and increased risk of eosinophilic inflammation at exacerbation (OR 9.16; p<0.001). Eosinophils ≥2% at exacerbation and eosinophil predominance at stable visits were associated with a lower risk of bacterial presence at exacerbation (OR 0.49; p=0.049 and OR 0.25; p=0.065, respectively). Bacterial infection at exacerbation was highly seasonal (winter versus summer OR 4.74; p=0.011) in predominantly eosinophilic patients.Eosinophilic inflammation is a common and stable phenotype in COPD. Blood eosinophil counts in the stable state can predict the nature of inflammation at future exacerbations, which when combined with an understanding of seasonal variation provides the basis for the development of new treatment paradigms for this important condition.

A novel lung explant model for the ex vivo study of efficacy and mechanisms of anti-influenza drugs.

Influenza A virus causes considerable morbidity and mortality largely because of a lack of effective antiviral drugs. Viral neuraminidase inhibitors, which inhibit viral release from the infected cell, are currently the only approved drugs for influenza, but have recently been shown to be less effective than previously thought. Growing resistance to therapies that target viral proteins has led to increased urgency in the search for novel anti-influenza compounds. However, discovery and development of new drugs have been restricted because of differences in susceptibility to influenza between animal models and humans and a lack of translation between cell culture and in vivo measures of efficacy. To circumvent these limitations, we developed an experimental approach based on ex vivo infection of human bronchial tissue explants and optimized a method of flow cytometric analysis to directly quantify infection rates in bronchial epithelial tissues. This allowed testing of the effectiveness of TVB024, a vATPase inhibitor that inhibits viral replication rather than virus release, and to compare efficacy with the current frontline neuraminidase inhibitor, oseltamivir. The study showed that the vATPase inhibitor completely abrogated epithelial cell infection, virus shedding, and the associated induction of proinflammatory mediators, whereas oseltamivir was only partially effective at reducing these mediators and ineffective against innate responses. We propose, therefore, that this explant model could be used to predict the efficacy of novel anti-influenza compounds targeting diverse stages of the viral replication cycle, thereby complementing animal models and facilitating progression of new drugs into clinical trials.

Steroid-induced Deficiency of Mucosal-associated Invariant T Cells in the Chronic Obstructive Pulmonary Disease Lung. Implications for Nontypeable Haemophilus influenzae Infection.

RATIONALE: Mucosal-associated invariant T (MAIT) cells are a recently described abundant, proinflammatory T-cell subset with unknown roles in pulmonary immunity. Nontypeable Haemophilus influenzae (NTHi) is the leading bacterial pathogen during chronic obstructive pulmonary disease (COPD) exacerbations and is a plausible target for MAIT cells. OBJECTIVES: To investigate whether MAIT cells respond to NTHi and the effects of inhaled corticosteroids (ICS) on their frequency and function in COPD. METHODS: Eleven subjects with COPD receiving ICS, 8 steroid-naive subjects with COPD, and 21 healthy control subjects underwent phlebotomy, sputum induction, bronchoalveolar lavage, and endobronchial biopsy. Pulmonary and monocyte-derived macrophages were cultured in vitro with NTHi. MEASUREMENTS AND MAIN RESULTS: Frequencies of Vα7.2+CD161+ MAIT cells, surface expression of the major histocompatibility complex-related protein 1 (MR1), and intracellular IFN-γ expression were measured by flow cytometry. MAIT-cell frequencies were reduced in peripheral blood of ICS-treated subjects with COPD (median 0.38%; interquartile range [IQR], 0.25-0.96) compared with healthy control subjects (1.8%; IQR, 1.4-2.5; P = 0.001) or steroid-naive patients with COPD (1.8%; IQR, 1.2-2.3; P = 0.04). MAIT cells were reduced in bronchial biopsies from subjects with COPD treated with steroids (0.73%; IQR, 0.46-1.3) compared with healthy control subjects (4.0%; IQR, 1.6-5.0; P = 0.02). Coculture of live NTHi increased macrophage surface expression of MR1 and induced IFN-γ from CD4 cells and CD8 cells, but most potently from MAIT cells (median IFN-γ-positive frequencies, 2.9, 8.6, and 27.6%, respectively). In vitro fluticasone and budesonide reduced MR1 surface expression twofold and decreased NTHi-induced IFN-γ secretion eightfold. CONCLUSIONS: MAIT cells are deficient in blood and bronchial tissue in steroid-treated, but not steroid-naive, COPD. NTHi constitutes a target for pulmonary MAIT-cell immune responses, which are significantly impaired by corticosteroids.

Dysregulation of Antiviral Function of CD8(+) T Cells in the Chronic Obstructive Pulmonary Disease Lung. Role of the PD-1-PD-L1 Axis.

RATIONALE: Patients with chronic obstructive pulmonary disease (COPD) are susceptible to respiratory viral infections that cause exacerbations. The mechanisms underlying this susceptibility are not understood. Effectors of the adaptive immune response-CD8(+) T cells that clear viral infections-are present in increased numbers in the lungs of patients with COPD, but they fail to protect against infection and may contribute to the immunopathology of the disease. OBJECTIVES: CD8(+) function and signaling through the programmed cell death protein (PD)-1 exhaustion pathway were investigated as a potential key mechanism of viral exacerbation of the COPD lung. METHODS: Tissue from control subjects and patients with COPD undergoing lung resection was infected with live influenza virus ex vivo. Viral infection and expression of lung cell markers were analyzed 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 in 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 up-regulated T-cell PD-1 expression in control and COPD samples. Concurrently, influenza significantly up-regulated the marker of cytotoxic degranulation (CD107a) on CD8(+) T cells (P = 0.03) from control subjects but not on those from patients with COPD. Virus-induced expression of the ligand PD-L1 was decreased on COPD macrophages (P = 0.04) with a corresponding increase in IFN-γ release from infected COPD explants compared with 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.