Samantha Thulborn

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; P<0.01), but not FEV1 or health status. There was a weak correlation between PTX3 and bacterial load (CFU: r=0.29, P<0.01; 16S qPCR: r=0.18, P=0.05). PTX3 was a poor predictor of bacterial colonization (defined as >105 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.

S45 Evaluating the sensitivity and specificity of active neutrophil elastase as a biomarker for bacterial infection in subjects with copd

Introduction COPD is a neutrophilic disease, with the majority of subjects having a sputum neutrophil percentage of >60%. Neutrophil elastase (NE) is a serine proteinase, secreted by neutrophils and macrophages during inflammation and has a role in the destruction of bacteria within the host. New advancements now allow accurate assessment of active protease levels in complex biological samples. We sought to investigate if active NE could be used as a biomarker for bacterial infection in subjects with COPD. Methods NE was quantified using ProteaseTagTM active NE Immunoassay (ProAxsis, Belfast) from cell-free sputum supernatant from 31 COPD subjects (20 Males; mean age 65, range 45 to 81) at stable state and during an exacerbation. Bacterial infection was defined as ≥107 CFU/mL in sputum. Subject demographics, sputum cell differential counts and polymerase chain reaction (PCR) for respiratory pathogens were measured. Results Active NE was higher during an exacerbation compared to stable state (fold difference (95% CI) 0.50 (0.22 to 0.78), p = 0.001) (Figure 1). NE correlated with total sputum neutrophils (p < 0.0001, r = 0.48) and total bacterial load measured by CFU/mL (p < 0.01, r = 0.39) and qPCR (p < 0.05, r = 0.33). When looking at the main respiratory pathogens no correlations were seen between H. influenzae (p = 0.43, r = −0.11), S. aureus (p = 0.34, r = −0.14) or S. pneumonia (p = 0.11, r = 0.23); however a correlation was seen between NE and M. catarrhalis (p = 0.01, r = 0.36). NE has an area under the receiver operator curve of 0.72 [0.58 to 0.85] to identify a bacterial infection with a sensitivity and specificity of 67.74% and 67.86% at a NE cut off of 2335ng/mL. Conclusion Active NE is elevated during a COPD exacerbation compared to baseline. Active NE is associated with neutrophilic inflammation and bacteria; and may be a viable biomarker for bacterial infection in COPD. Abstract S45 Figure 1 Sputum active NE levels at stable and exacerbation state from 31 paired COPD subjects. Mean and 95% CI

Type-2 CD8+ T lymphocytes responsive to PGD2 and LTE4 in severe eosinophilic asthma

The functions and in vivo roles of type-2 CD8+ T cells in humans have not been well defined and this cell type has been largely overlooked in models of disease. We investigated this in the context of severe asthma with persistent airway eosinophilia - a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

Synergistic activation of pro-inflammatory type-2 CD8 + T lymphocytes by lipid mediators in severe eosinophilic asthma

Human type-2 CD8+ T cells are a cell population with potentially important roles in allergic disease. We investigated this in the context of severe asthma with persistent airway eosinophilia—a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines, which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

P114 Glucocorticoid receptor α and β expression in bronchial epithelial cells infected with nthi

Introduction Steroids act through the glucocorticoid receptor (GR), of which the alpha-isoform (GRα) is most abundant. Neutrophilic COPD is associated both with resistance to steroids and with airway bacterial infection, most commonly by non-typeable Haemophilus influenzae (NTHi). We hypothesised that NTHi downregulates GRα or upregulates its inhibitory beta-isoform (GRβ) in bronchial epithelial cells, thereby inhibiting their response to steroids. Method Bronchial epithelial cell line Beas-2B were treated with the corticosteroid Fluticasone propionate (0, 1 nM or 100 nM) for 2 hours prior to infection with 1.5 × 104, 1 × 106 and 1.5 × 107 CFU/ml NTHi (low, medium and high load respectively). 6 hours post-infection supernatants were collected and RNA extracted from cells. RNA was reverse transcribed to cDNA in which levels of GRα, GRβ and GAPDH were determined by SYBR Green PCR and expression calculated using the Pfaffl method relative to untreated cells. Results GRα expression in Beas-2B was enhanced by corticosteroid treatment in a stepwise manner for 1 nM (median fold increase from untreated: 1.491, IQR: 1.305–1.668), and 100 nM (median: 1.742 fold, IQR: 1.51–1.9) (p≥0.05 for both). Increasing load of NTHi showed no effect on GRα expression. GRβ expression showed little fluctuation from levels of untreated cells upon infection with NTHi alone or high dose corticosteroid, however the two showed a trend to synergistically decrease in GRβ expression when treated with high corticosteroid and high load of NTHi (median fold change from untreated cells: 0.542 (IQR:0.453–0.578) (p=0.25) (figure 1). Abstract P114 Figure 1 Expression of GRβ in Beas-2B cells relative to untreated cells normalised to GAPDH. Conclusions Corticosteroid treatment shows a trend to increased GRα expression on Beas-2B cells. Increased NTHi load has no effect on GRα expression of Beas-2B cells. GRβ expression appears not to be affected by NTHi infection alone, however with corticosteroid shows a trend to decreased expression. The experiments are to be repeated in primary bronchial epithelial cells to determine whether they follow the trend seen here in a cell line.

P113 Antimicrobial peptides in inflammatory phenotypes of copd

Introduction Antimicrobial peptides act to defend the host from microbial action and colonisation. Patients with COPD and neutrophilic inflammation experience bacterial colonisation more frequently than other COPD phenotypes. Here we assess the levels of five antimicrobial peptides in peripheral blood and sputum in relation to their inflammatory phenotype. We hypothesise that patients with neutrophilic inflammation have lower antimicrobial peptide levels than other COPD inflammatory phenotypes and that the presence of non-typeable haemophilus influenzae (NTHi) is associated with low antimicrobial peptide levels. Method Plasma and sputum supernatants from 8 healthy donors, 18 COPD patients and 10 non-eosinophilic asthmatics were tested for SLPI, osteopontin, lysozyme, elafin and beta defensin-1 by ELISA. Patients were stratified into eosinophilic and neutrophilic groups with a 3% sputum eosinophil cut-off. NTHi was measured in sputum plugs by qPCR of the Omp P6 gene. Results Levels of antimicrobial peptides in plasma and sputum showed no difference between those with eosinophilic and neutrophilic COPD. Between disease groups, beta defensin-1 levels are higher in plasma of COPD patients (median: 10.92 ng/ml (IQR: 4.137–18.09)) than healthy individuals (median: 3.665 ng/ml (IQR: 2.59–4.549), p=0.0033) and non-eosinophilic asthmatics (median: 4.984 ng/ml IQR: 3.334–7.208), p=0.0442) (figure 1). No antimicrobial peptide correlated with NTHi levels in the sputum plug. Abstract P113 Figure 1 Concentrations of beta defensin-1 in plasma from healthy individuals, asthmatics and COPD patients. Conclusions Similar levels of SLPI, osteopontin, lysozyme, elafin and beta defensin-1 in sputum and plasma between COPD phenotypes suggests that defence against pathogens by these antimicrobial peptides is not lacking in differential inflammatory COPD phenotypes. The role antimicrobial peptides play in NTHi colonisation remains to be determined.

S116 Cell-dissociated haemophilus influenzae and bacteria-associated inflammatory mediators in the airways of patients with chronic obstructive pulmonary disease

Background Patients with COPD have a susceptibility to respiratory tract infections associated with increased pulmonary inflammation. Bacteria can reside within the host as cell-associated (attached to host cells via adhesins, pili or biofilm formation) or cell-dissociated bacteria. It is unclear how bacteria-to-cell interactions affect pulmonary inflammation and whether these levels differ over an exacerbation time course. We sought to investigate the effects of Haemophilius influenzae cell-interaction upon airway inflammation and whether the levels of H. influenzae bacteria and cell-dissociated bacteria differ over an exacerbation time course. Methods Cell differential counts were carried out on sputum samples as per standard protocol. Bacterial DNA was extracted and H.influenzae was quantified using qPCR from the sputum plug (contains cell-associated and dissociated bacteria) and the sputum cell-free supernatant (cell-dissociated bacteria only). Inflammatory mediators (IL-1α, TNF-α, IL-8 and neutrophil elastase (NE)) were measured in the sputum supernatant using commercial assays. Results 63 patients (77% male; average age of 69 (45–88); FEV1 percentage predicted of 53%; mean percentage neutrophil count in sputum of 65%) at stable state were analysed. Levels of H. influenzae in the supernatant only correlated with the sputum total cell count (r=0.38; p=0.03). Levels of H. influenzae in the plug correlated with inflammatory mediators (sputum neutrophil percentage r=0.42, p=0.01; sputum macrophage percentage r=−0.35, p=0.04; IL-1α r=0.36, p=0.03; IL-8 r=0.49, p<0.01; NE r=0.40, p=0.02). The exacerbation time course in 10 paired COPD subjects was examined. There was no significant difference in H. influenzae levels in the plug (p=0.89) (figure 1A). However, there was a significant increase in levels in the supernatant over the exacerbation time course (p=0.05) (figure 1B). Conclusion H. influenzae levels in the sputum plug appear to have much more of an effect on airway inflammation than levels of cell-dissociated H. influenzae suggesting that cell-associated bacteria may be a driver of airway inflammation in COPD. Further investigation into this highly complicated relationship needs to be conducted. Abstract S116 Figure 1 Levels of H. influenzae in the sputum plug (A) and in the sputum supernatant (B) during an exacerbation time course in 10 paired subjects with COPD.

P234 Sputum cytokines and clinical biomarkers in severe asthma

Introduction Emerging treatments for type-2 high asthma such as anti-IL-5 (mepolizumab) and anti-IL-4 and IL-13 (dupilumab) target specific cytokine pathways resulting in type-2 inflammation. Whether patients with type 2 inflammation respond equally to both treatment or have distinct IL-13 and IL-5 profiles is currently unclear. We have tested the hypothesis that these pathways may function independently of each other and that simple biomarkers can help differentiate IL-13 and IL-5 high patients. Methods Patients with well characterised, severe asthma were evaluated with the blood eosinophil count and fractional exhaled nitric oxide (FeNO). Patients also had paired measurements of type-2 cytokines in induced sputum samples. Sputum cytokines were measured using a Luminex assay. Results We found that there was no relationship between the blood eosinophil count and FeNO. There was a positive correlation between FeNO and sputum IL-13 (r = 0.51, p < 0.01) and blood eosinophils and sputum IL-5 (r = 0.47, p < 0.01). Conclusions These findings suggest that readily available, non-invasive biomarkers may be able to differentiate sub-phenotypes in type-2 high asthma. Post-hoc analysis of clinical trial data of anti-IL-5 and anti-IL-4 and IL-13 treatments based on the predominant clinical biomarker would be of interest to see if these predict response to treatment. Simple biomarkers may be of use in deciding which of the emerging biological treatments to use in severe, type-2 high asthma.

S96 Free-living Haemophilus Influenzae is associated with increased pulmonary inflammation

Introduction The most common pathogen in the lower airway of patients with COPD is Haemophilus influenzae. H. influenzae has been shown to be linked to inflammation and increased inflammation. Emerging evidence shows that pathogens can exist as either cell-associated or free-living (non-cell associated). We investigated whether detectable free-living H. influenzae correlates with pulmonary inflammation. Methods Cell-free sputum supernatants samples from 29 COPD patients (24 men), with a mean (range) age of 71 (45 to 88) years were analysed. All samples were collected at stable state and bacterial DNA was extracted, using a commercial assay and then quantified using real time-PCR utilising taqman hydrolysis probes. The omp P6 gene from H. influenzae was inserted into a positive cloning vector and transformed to generate plasmids. These plasmids were used as standards within the qPCR, allowing the accurate detection of very small levels of H. influenzae within the samples. Cytokines were measured using the meso-scale multi-array platform within the same sample set. Results Free Living H. influenzae was detected in 15/29 (52%) of cell-free samples, with a bacterial load of (geometric mean (95% CI)) of 1.23 × 106 gene copies/ml (2.63 × 105 to 5.75 × 106). Correlations were seen between free-living H. influenzae and Interleukin-1 Beta (IL1-β) (r = 0.47, p = 0.01), MMP8 (p = 0.04, r = 0.38), CCL3 (p = 0.002 =, r = 0.57), CCL13 (p = 0.02, r = 0.54), CCL26 (p = 0.03, r = -0.31), CCL4 (p = 0.04, r = 0.38). No significant correlations were seen between free-living H. influenzae and IL8 (p = 0.11, r = -0.30), IL10 (p = 0.10, r = -0.36) or TNF-alpha (p = 0.61, r = -0.12). Conclusion Free-living H. influenzae is associated with increased pro-inflammatory mediators in the airway. Whether this is related to the pathogenesis of COPD needs to be further investigated.

S95 Peripheral blood CRTH2 positive cell count in patients with severe eosinophilic asthma

Background CRTH2 antagonism has been shown to reduce eosinophilic airway inflammation and improve lung function in patients with severe eosinophilic asthma. To better understand the role of CRTH2 in the pathogenesis of this asthma phenotype, we have carried out a cross-sectional study to investigate the CRTH2 positive cell counts in peripheral blood of patients with severe eosinophilic asthma. Methods Blood was taken from 12 controls and 33 patients with asthma, 21 of whom met the 2014 ERS/ATS guideline criteria for severe asthma and had historical evidence of eosinophilic airway inflammation as defined before (Pavord et al. Lancet 2012;380:651–9). Th2 were detected as CD3+CD4+CRTH2+, Tc2 as CD3+CD8+CRTH2+, eosinophils as SSChighCRTH2+, and basophils as CD123+CRTH2+ by flow cytometry, and numbers presented as total cell counts in peripheral blood. Data were analysed using one-way ANOVA followed by the Newman-Keuls test. Results CRTH2 cell counts were reasonably repeatable within patients (ICC 0.84; n = 9). Mean ± SD CRTH2+ cell counts were 168 ± 81, 322 ± 191, 710 ± 322 and 290 ± 179 × 106 cells/L in normal controls (n = 12), patients with mild to moderate asthma (n = 12), patients with severe asthma at BTS step 4 (n = 10), and patients with severe asthma at BTS step 5 (n = 11) respectively (Figure 1). Most CRTH2 + cells were eosinophils (Figure 1).Abstract S95 Figure 1 Comparison of eosinophil, basophil, Th2, Tc2 and total CRTH2+ cell counts in the blood from healthy control and different asthma patients. (p < 0.0001 for CRTH2+ and eosinophils; p < 0.001 for basophils; p < 0.05 for Th2; and p < 0.1 for Tc2. * p < 0.05 between the indicated groups) Conclusion Blood CRTH2+ cells are increased in subjects with severe eosinophilic asthma, mainly because of increased CRTH2+ eosinophils. Eosinophils and basophils numbers are significantly increased in severe eosinophilic asthma at step 4 but not step 5. Th2 and Tc2 cell numbers are less clearly associated with severe asthma. CRTH2+ cell numbers are lower in patients treated with prednisolone.