Kasim Jiwa

Telomere Dysfunction and Senescence-associated Pathways in Bronchiectasis

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Profiling inflammation and tissue injury markers in perfusate and bronchoalveolar lavage fluid during human ex vivo lung perfusion

Abstract OBJECTIVES: Availability of donor lungs suitable for transplant falls short of current demand and contributes to waiting list mortality. Ex vivo lung perfusion (EVLP) offers the opportunity to objectively assess and recondition organs unsuitable for immediate transplant. Identifying robust biomarkers that can stratify donor lungs during EVLP to use or non-use or for specific interventions could further improve its clinical impact. METHODS: In this pilot study, 16 consecutive donor lungs unsuitable for immediate transplant were assessed by EVLP. Key inflammatory mediators and tissue injury markers were measured in serial perfusate samples collected hourly and in bronchoalveolar lavage fluid (BALF) collected before and after EVLP. Levels were compared between donor lungs that met criteria for transplant and those that did not. RESULTS: Seven of the 16 donor lungs (44%) improved during EVLP and were transplanted with uniformly good outcomes. Tissue and vascular injury markers lactate dehydrogenase, HMGB-1 and Syndecan-1 were significantly lower in perfusate from transplanted lungs. A model combining IL-1β and IL-8 concentrations in perfusate could predict final EVLP outcome after 2u2009h assessment. In addition, perfusate IL-1β concentrations showed an inverse correlation to recipient oxygenation 24u2009h post-transplant. CONCLUSIONS: This study confirms the feasibility of using inflammation and tissue injury markers in perfusate and BALF to identify donor lungs most likely to improve for successful transplant during clinical EVLP. These results support examining this issue in a larger study.

The role of interleukin-1β as a predictive biomarker and potential therapeutic target during clinical ex-vivo lung perfusion

Background Extended criteria donor lungs deemed unsuitable for immediate transplantation can be reconditioned using ex vivo lung perfusion (EVLP). Objective identification of which donor lungs can be successfully reconditioned and will function well post-operatively has not been established. This study assessed the predictive value of markers of inflammation and tissue injury in donor lungs undergoing EVLP as part of the DEVELOP-UK study. Methods Longitudinal samples of perfusate, bronchoalveolar lavage, and tissue from 42 human donor lungs undergoing clinical EVLP assessments were analyzed for markers of inflammation and tissue injury. Levels were compared according to EVLP success and post-transplant outcomes. Neutrophil adhesion to human pulmonary microvascular endothelial cells (HPMECs) conditioned with perfusates from EVLP assessments was investigated on a microfluidic platform. Results The most effective markers to differentiate between in-hospital survival and non-survival post-transplant were perfusate interleukin (IL)-1β (area under the curve = 1.00, p = 0.002) and tumor necrosis factor-α (area under the curve = 0.95, p = 0.006) after 30 minutes of EVLP. IL-1β levels in perfusate correlated with upregulation of intracellular adhesion molecule-1 in donor lung vasculature (R2 = 0.68, p < 0.001) and to a lesser degree upregulation of intracellular adhesion molecule-1 (R2 = 0.30, p = 0.001) and E-selectin (R2 = 0.29, p = 0.001) in conditioned HPMECs and neutrophil adhesion to conditioned HPMECs (R2 = 0.33, p < 0.001). Neutralization of IL-1β in perfusate effectively inhibited neutrophil adhesion to conditioned HPMECs (91% reduction, p = 0.002). Conclusions Donor lungs develop a detectable and discriminatory pro-inflammatory signature in perfusate during EVLP. Blocking the IL-1β pathway during EVLP may reduce endothelial activation and subsequent neutrophil adhesion on reperfusion; this requires further investigation in vivo.

Excess Mucin Impairs Subglottic Epithelial Host Defense in Mechanically Ventilated Patients

&NA; Rationale: Aspiration of infective subglottic secretions causes ventilator‐associated pneumonia (VAP) in mechanically ventilated patients. Mechanisms underlying subglottic colonization in critical illness have not been defined, limiting strategies for targeted prevention of VAP. Objectives: To characterize subglottic host defense dysfunction in mechanically ventilated patients in the ICU; to determine whether subglottic mucin contributes to neutrophil phagocytic impairment and bacterial growth. Methods: Prospective subglottic sampling in mechanically ventilated patients (intubated for four or more days), and newly intubated control patients (intubated for less than 30 min); isolation and culture of primary subglottic epithelial cells from control patients; laboratory analysis of host innate immune defenses. Measurements and Main Results: Twenty‐four patients in the ICU and 27 newly intubated control patients were studied. Subglottic ICU samples had significantly reduced microbiological diversity and contained potential respiratory pathogens. The subglottic microenvironment in the ICU was characterized by neutrophilic inflammation, significantly increased proinflammatory cytokines and neutrophil proteases, and altered physical properties of subglottic secretions, including accumulation of mucins. Subglottic mucin from ICU patients impaired the capacity of neutrophils to phagocytose and kill bacteria. Phagocytic impairment was reversible on treatment with a mucolytic agent. Subglottic mucus promoted growth and invasion of bacterial pathogens in a novel air‐liquid interface model of primary human subglottic epithelium. Conclusions: Mechanical ventilation in the ICU is characterized by substantial mucin secretion and neutrophilic inflammation. Mucin impairs neutrophil function and promotes bacterial growth. Mucolytic agents reverse mucin‐mediated neutrophil dysfunction. Enhanced mucus disruption and removal has potential to augment preventive benefits of subglottic drainage.

S84 Sputum neutrophils but not Interleukin-8 (IL-8) or Interleukin 17 (IL-17) correlate with the Bronchiectasis Severity Index (BSI)

Background Bronchiectasis is a progressive neutrophilic inflammatory lung disease associated with abnormal local cytokine release with possible systemic overspill. Early data suggests that interleukin-17 (IL-17) could be involved in the enhanced infiltration of neutrophils in the lungs, via the induction of IL-8 release, and has emerged as a possible biomarker for other chronic neutrophilic lung diseases. Aims to investigate the potential use of IL-17 and IL-8 as biomarkers of disease severity in bronchiectasis by utilising a multidimensional clinical severity scoring system, the Bronchiectasis Severity Index (BSI). correlate sputum neutrophils and pathogen status with serum or sputum IL-17 and IL-8 levels. Methods Spontaneous sputa and sera were collected from stable adult bronchiectasis patients attending a specialist clinic. We quantified both IL-17 and IL -8 concentrations in the pulmonary compartment (sputum) and the systemic compartment (serum) of 119 stable bronchiectasis patients and 26 healthy volunteers. Sputum neutrophils were conducted using standard methods. Results The mean patient age was 65 years, with 24% in mild BSI, 39% moderate BSI and 46% (43% idiopathic, 24% post infectious). IL-17 in the sputum of bronchiectasis patients was found to be two-fold greater than in serum suggesting “local” release (10 pg/ml vs 5 pg/ml). Statistical analysis revealed a significant correlation between these two variables, suggesting a “spillover” of cytokines from the lungs (p < 0.001). However, there was no significant difference in serum IL-17 levels between bronchiectasis and healthy subjects (0 ± 2 pg/ml). In addition, no significant correlation was found between IL-8 and IL-17 levels in the sputum of patients. Sputum IL-17 levels were found to have a significant negative correlation with BSI severity scoring, but this was not reproduced when individual BSI parameters were analysed. IL-8 similarly performed poorly in correlating with BSI. In contrast more severe BSI scores were significantly associated with higher% neutrophils in sputa (p = 0.045). Conclusions The clinical utility of IL-17 and IL-8 as biomarkers for the prediction of disease severity in bronchiectasis patients appears poor. These data may also suggest targeting these chemokines are of limited value. Focus in bronchiectasis may need shifted from neutrophil chemokines to factors that inhibit apoptosis and/or promote neutrophil persistence in the airway.

S136 Potential therapeutic benefits of the human amniotic epithelium cell secretome during ex-vivo perfusion of donor lungs

Introduction Ex-vivo lung perfusion (EVLP) is used to assess and potentially recondition donor lungs that are not initially suitable for transplantation. In a recent UK study, EVLP was associated with higher primary graft dysfunction (PGD) rates compared to standard lung evaluation procedure. PGD is a process characterised by leucocyte-endothelial adhesion and acute lung injury. In this study, donor lung leucocyte content and endothelial activation before and after EVLP was assessed and then the potential of the human amnion epithelial cell (hAEC) secretome to reduce endothelial cell – leucocyte interaction was evaluated. Methods Donor lung tissue, pre and post EVLP (n = 16), was stained with CD45, neutrophil elastase and IL-1 receptor type I antibodies to assess endothelial activation, and leukocyte recruitment. Human umbilical vein endothelial cells (HUVECs) and human amnion epithelial cells were isolated from term (n = 5) and preterm (n = 5) placentas. HUVECs activated with IL-1β were used for leucocyte rolling and adhesion studies in an in-vitro model. The effects of hAEC-conditioned media (hAEC secretome) on leucocyte rolling, adhesion and HUVEC expression of adhesion molecules (E-selectin, ICAM-1, and VCAM) was assessed. Results CD45 positive cells in the donor lung vasculature significantly decreased during EVLP (p = 0.028). Neutrophil elastase positive cells were significantly lower in accepted donor lungs (n = 4) compared to declined lungs (n = 4) (p = 0.0044). However, IL-1 Receptor Type 1 expression significantly increased after EVLP (p = 0.048). The hAEC secretome derived from both term and pre-term placentas significantly reduced endothelial ICAM-1 levels, p = 0.013 and p = 0.0085 respectively. This was accompanied by a significant decrease in leucocyte rolling and leucocyte-endothelial adhesion, with both term (p = 0.041) and preterm (p = 0.014) hAEC secretome. Abstract S136 Figure 1 Leukocyte adhesion to HUVECs stimulated with 5ng/ml IL-1β; untreated control, treated with human amnion epithelial cell (hAEC) term conditioned media (T-CM), and hAEC preterm conditioned media (PT-CM) Discussion EVLP reduces the intravascular leucocyte content of the donor lung, probably due to the presence of a leucocyte filter in the perfusion circuit. However, the endothelium remains primed to activation with IL-1 receptor expression increasing during EVLP. The secreted products of hAECs reduce endothelial activation and limit leukocyte-endothelial interactions. Further studies are required in a suitable in-vivo model to determine if hAECs, or their secretome, are a potential therapeutic option during EVLP to protect against primary graft dysfunction after reperfusion of the transplanted lung.

S46 Neutrophil vascular endothelial growth factor (VEGF) as a driving force for angiogenesis in bronchiectasis

Introduction Bronchiectasis (BR) in a pulmonary disease thought to involve a characteristic dilation of the bronchi resulting from a cycle of airway infection and inflammation. This inflammation is believed to be driven by neutrophils, which are present in the BR lung in high number. Vascular endothelial growth factor (VEGF) is a pro-angiogenic cytokine that may be upregulated in BR and could contribute towards creating a pro-angiogenic airway environment by supporting neutrophil migration into the airway tissue, however this has yet to be shown. Aims 1) Examine the BR airway for any indications of increased angiogenesis, 2) Assess the ability of neutrophils to secrete VEGF upon stimulation in vitro, 3) Evaluate sera/sputa samples VEGF concentration to determine if VEGF could act as a biomarker for BR severity. Methods Healthy volunteer (HV) and BR endobronchial biopsies were stained with a HRP conjugated anti-CD31 antibody, allowing blood vessels to be counted in a blinded manner. Peripheral blood neutrophils isolated from HV were stimulated (e.g. with TNF-α or bacterial PAMPs) for 4 hours, VEGF levels in supernatants were then quantified using ELISA. A VEGF ELISA was also used to determine VEGF concentration in sera and sputa samples from BR patients (n = 115), categorised by bronchiectasis severity index (BSI) scores and sera samples from HV controls (n = 26) Results Endobronchial biopsies from BR airways had a significantly (p < 0.05) higher number of blood vessels per mm of basement membrane than HV samples (18 and 9 blood vessels/mm basement membrane respectively). Stimulation of HV neutrophils with a variety of molecules (PMA, fMLP, LPS, TNF-α etc.) resulted in a significant increase in VEGF secretion compared to unstimulated (p < 0.05). Although elevated VEGF was found in some patient samples there was no significant correlation between sera/sputa VEGF and individual patient BSI scores. Conclusion The increased presence of vascular tissue seen in BR could indicate a pro-angiogenic airway environment in BR. The in vitro data collected also show that a variety of stimulants can initiate secretion of VEGF by neutrophils. However, our data does not suggest that VEGF levels in sera or sputa can be used to predict disease severity.

S65 Large and small airway epithelial cell senescence present in COPD and bronchiectasis

Introduction and Objectives Accelerated lung ageing has been implicated in the pathogenesis of COPD and therefore targeting cellular senescence may have therapeutic benefit. COPD is increasingly felt to have significant sub-phenotypes with large and small airway involvement. The airway epithelium likely endures the majority of potentially senescence-inducing insults. However, data on airway epithelial cell (AEC) senescence in COPD is limited and comparisons between large and small airways are lacking. Furthermore, the role of infection in COPD-associated senescence is unclear. To date, senescence in bronchiectasis has not been investigated as a model for infection-induced senescence. We sought to determine AEC expression of senescence-associated markers in COPD and bronchiectasis and to compare large and small airways. Methods Lung explant tissue from our transplant programme from COPD (n = 19) and bronchiectasis (n = 14) with resection tissue from smokers without lung disease (control) (n = 11) was stained for senescence-associated markers by immunohistochemistry. Staining was quantified semi-quantitatively. Fluorescence in situ hybridisation (FISH) was used to investigate telomere length and possible co-localisation with DNA damage-associated proteins. Abstract S65 Figure 1. Expression of Ki67 (A), SIRT1 (B), p16 (C) and γH2AX (D) in large AECs and small AECs (E, F, G and H, respectively). Results are expressed as mean ± SEM. Statistics: Kruskal-Wallis and Mann-Whitney U test. * p < 0.05, ** p < 0.01, *** p < 0.001. Results In large AECs, expression of the proliferation marker ki67 and the anti-ageing protein sirtuin 1 (SIRT1) was decreased in COPD as compared to bronchiectasis and controls. There was no difference in expression of the cell-cycle inhibitor p16 and the DNA damage associated foci ?H2AX between the three groups. In small AECs, SIRT1 was decreased in COPD compared to controls and p16 and ?H2AX were increased. Here, ki67 expression did not differ between groups. In bronchiectasis, there was no significant change in senescence marker expression compared to controls, with the exception of decreased SIRT1 in large AECs. Marker expression was not significantly correlated with FEV1 or smoking history. Preliminary work suggests potential co-localisation of ?H2AX at telomeres with ongoing analysis underway. Conclusions Differential expression of senescence-associated markers between large and small airways in COPD may reflect the distinct patterns of inflammation and functional impairment occurring in the two airway compartments. There is some evidence suggesting a role for senescence in bronchiectasis, though this is less clear than for COPD. Further markers need to be investigated.