The Use of Biomarkers to Explore Inflammation and Phenotypes during Exacerbations of Chronic Obstructive Pulmonary Disease

Exacerbations of chronic obstructive pulmonary disease (COPD) are heterogeneous. In particular the association of airway inflammation with pathogens during exacerbations of COPD remains largely unknown. The identification of biomarkers of COPD aims to characterise and phenotype COPD and COPD exacerbations, explore mechanisms of pathogenesis and provide decisions regarding targeted treatment strategies. After validating the Meso scale discovery platform, biomarkers in stable state and during exacerbations COPD were explored. COPD exacerbations were associated with increased systemic and pulmonary inflammation and exacerbations associated with bacteria, virus and sputum eosinophils could be identified by sensitive and specific biomarkers. Multivariate statistical analysis identified independent biological exacerbations phenotypes and that these phenotypes could be predicted from stable state biomarkers. Finally a biomarker directed strategy using the peripheral blood eosinophil count to guide systemic corticosteroid therapy during exacerbations of COPD was effective, safe and identified phenotypes which have particular prognostic and therapeutic characteristics. To conclude, biomarkers can define the heterogeneity of COPD and COPD exacerbations. The identification of COPD phenotypes can potentially move the management towards phenotype specific management and personalised medicine

Investigating the role of pentraxin 3 as a biomarker for bacterial infection in subjects with COPD

BACKGROUND: Pentraxin 3 (PTX3) is an acute phase protein, involved in antibacterial resistance. Recent studies have shown PTX3 levels to be elevated in the presence of a bacterial infection and in a murine sepsis model. OBJECTIVE: We aim to investigate if sputum PTX3 can be used as a biomarker for bacterial infection in subjects with COPD. MATERIALS AND METHODS: Sputum samples from 142 COPD patients (102 men) with a mean (range) age of 69 years (45-85) and mean (SD) post-bronchodilator percentage predicted forced expiratory volume in 1 second (FEV1) of 50% (19) were analyzed for PTX3, using a commercial assay at stable state and during an exacerbation. Association with bacteria, from culture, quantitative real-time polymerase chain reaction (qPCR) and colony-forming units (CFU) was investigated. RESULTS: The geometric mean (95% CI) PTX3 level at stable state was 50.5 ng/mL (41.4-61.7). PTX3 levels correlated with absolute neutrophil count in sputum (r=0.37; P10(5) CFU/mL at stable state) with a receiver-operating characteristic (ROC) area under the curve (AUC) of 0.59 and 95% confidence interval (CI) 0.43-0.76 (P=0.21). During an exacerbation, there was a modest increase in PTX3 (fold difference 0.15, 95% of difference 0.02-0.29; P=0.02), and PTX3 fared better at identifying a bacteria-associated exacerbation (ROC AUC 0.65, 95% CI 0.52-0.78, P=0.03). CONCLUSION: PTX3 is associated with bacterial infection in patients with COPD, but its utility as a biomarker for identifying a bacteria-associated exacerbation warrants further studies

Impaired P2X1 Receptor–Mediated Adhesion in Eosinophils from Asthmatic Patients

Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at −60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP–evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin–dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration

Impaired P2X1 Receptor–Mediated Adhesion in Eosinophils from Asthmatic Patients

Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at −60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP–evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin–dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration

Microbiome balance in sputum determined by PCR stratifies COPD exacerbations and shows potential for selective use of antibiotics

BACKGROUND: While a subgroup of patients with exacerbations of chronic obstructive pulmonary disease (COPD) clearly benefit from antibiotics, their identification remains challenging. We hypothesised that selective assessment of the balance between the two dominant bacterial groups (Gammaproteobacteria (G) and Firmicutes (F)) in COPD sputum samples might reveal a subgroup with a bacterial community structure change at exacerbation that was restored to baseline on recovery and potentially reflects effective antibiotic treatment. METHODS: Phylogenetically specific 16S rRNA genes were determined by quantitative real time PCR to derive a G:F ratio in serial sputum samples from 66 extensively-phenotyped COPD exacerbation episodes. RESULTS: Cluster analysis based on Euclidean distance measures, generated across the 4 visit times (stable and exacerbation day: 0,14 and 42) for the 66 exacerbation episodes, revealed three subgroups designated HG, HF, and GF reflecting predominance or equivalence of the two target bacterial groups. While the other subgroups showed no change at exacerbation, the HG cluster (n = 20) was characterized by G:F ratios that increased significantly at exacerbation and returned to baseline on recovery (p<0.00001); ratios in the HG group also correlated positively with inflammatory markers and negatively with FEV1. At exacerbation G:F showed a significant receiver-operator-characteristic curve to identify the HG subgroup (AUC 0.90, p<0.0001). CONCLUSIONS: The G:F ratio at exacerbation can be determined on a timescale compatible with decisions regarding clinical management. We propose that the G:F ratio has potential for use as a biomarker enabling selective use of antibiotics in COPD exacerbations and hence warrants further clinical evaluation

Cluster analysis of qPCR-determined G:F ratios reveals three subgroups with different patterns of change through exacerbation.

<p><b>(A)</b> Heatmap representing the clustering of exacerbation episodes based on G:F ratio pattern across the four visit times. Blue shows Firmicute dominance and yellow Gammaproteobacterial dominance. <b>(B)</b> Changes in G:F across visit times. Mean ±SD. Points represent the individual sample G:F ratios.(****) p<0.0001. HF = High Firmicutes, HG = High Gammaproteobacteria and GF = Balanced G:F.</p

Comparison of inflammatory markers between the three clusters.

<p>Comparison of inflammatory markers between the three clusters.</p

A single G:F ratio assay at exacerbation identifies membership of the HG cluster.

<p>Abbreviations G—Gammaproteobacteria; <i>M</i>. <i>cat–M</i>. <i>catarrhalis</i>; <i>H</i>. <i>inf–H</i>. <i>influenzae</i>; F–Firmicutes; <i>S</i>. <i>pneum–S</i>. <i>pneumoniae</i>; nph—neutrophils; eos–eosinophils.</p

Comparison of patient characteristics between G:F clusters.

<p>Comparison of patient characteristics between G:F clusters.</p

Patient characteristics.

<p>Patient characteristics.</p