Lipoxin A4 on neutrophil reprogramming in bronchiectasis

INTRODUCTION Bronchiectasis is a common chronic debilitating respiratory condition. Patients suffer daily cough, excess sputum production and recurrent chest infections because of inflamed and permanently damaged airways. The pathogenesis of bronchiectasis is poorly understood. Pulmonary pathology shows excess neutrophilic airways inflammation, but despite this over two thirds of patients are chronically infected with potential pathogenic microorganisms. The acute inflammatory response is a protective mechanism that is evolved to eliminate invading organisms and should ideally be self-limiting and lead to complete resolution. The driver for persistent neutrophilic airway inflammation in bronchiectasis is unknown, but infection is considered to play a major role. AIMS The main aims of this thesis were to: (i) Characterize neutrophils in the serum and airways in bronchiectasis in the stable state and during exacerbations; (ii) Cohort study to establish if LXA4 deficiency correlates with disease severity (iii) Characterize lipids in bronchiectasis airways and peripheral blood to establish the correlation of LXA4 to disease severity; (iv) To investigate a potential mechanism for low levels of LXA4 in bronchiectasis, lipoxin biosynthetic genes expression will be measured; (v) Assess the anti-inflammatory and pro resolution effect of LXA4 on neutrophils and monocyte derived macrophages from healthy volunteers; (vi) Assess the anti-inflammatory and pro resolution effect of LXA4 on neutrophils during exacerbations in bronchiectasis and community acquired pneumonia. Methods (I) To establish the serum neutrophil subtype in stable state and following antibiotic treatment in patients with bronchiectasis, the following studies were done. Inclusion criteria: Patients aged 18-80 were recruited. All had an established radiological diagnosis of bronchiectasis (CT of the chest). Patients had clinically significant bronchiectasis, aetiology being either idiopathic or post infection. Exclusion Criteria: current smokers or ex-smokers of less than 1 year; >20 pack year history; cystic fibrosis; active allergic bronchopulmonary aspergillosis; active tuberculosis; poorly controlled asthma; severe COPD requiring nebulised bronchodilators or long term oxygen therapy; patients on aspirin or leukotriene inhibitors, pregnancy or breast feeding, active malignancy. A. 6 patients with mild bronchiectasis, 6 patients with severe bronchiectasis and 6 healthy volunteers were recruited. Serum and airways neutrophils were subsequently isolated. Neutrophil apoptosis, CD11b and CD62L expression, myeloperoxidase release, superoxide generation, phagocytosis and killing of GFP labeled bacteria were assessed. B. To compare serum with airways neutrophils function, bacterial phagocytosis and killing of GFP labeled bacteria was done, with both serum and airways neutrophils. Samples were obtained from the above group of patients. C. To establish neutrophil function following antibiotic treatment, 6 bronchiectasis patients at the beginning (day1) and the end (day14) of intravenous antibiotic therapy for an exacerbation were studied. As a control group, 6 community acquired pneumonia patients at the beginning (day1) and the end (day 5) of intravenous antibiotic therapy for infection were studied. Induced sputum and peripheral blood was taken at day1 and 5, where able. Phagocytosis and killing of GFP labeled bacteria was assessed and the two groups compared. (II) To address if lipoxin A4 deficiency correlates with disease severity, a cohort study was done in bronchiectasis patients. 169 patients were recruited and followed up for 1 year. Assessments done were Bronchiectasis severity index, systemic inflammatory markers (white cell count, ESR and c-reactive protein), Forced Expired Volume in 1sec, Forced Vital Capacity and its ratio, antibiotic courses in 1 year, hospital admissions in 1 year, sputum microbiology, quality of life assessments by Leicester Cough Questionnaire and St. Georges Respiratory Questionnaire, interleukin 8, myeloperoxidase, neutrophil elastase and leukotriene B4 (from sputum). (III) To assess effect of lipoxin on disease severity, 6 healthy volunteers, 10 patients with mild disease, 15 with moderate and 9 with severe disease were recruited. Disease severity was calculated as per the bronchiectasis severity index. All participants had 60mls of blood taken and underwent a bronchoscopy. Two segments of the lungs were washed out from bronchiectasis patients, an area affected by bronchiectasis and an area unaffected by bronchiectasis. This led to patients acting as their own internal control. Serum and airways neutrophils (from both segments) were subsequently isolated. Assessments done were systemic inflammatory markers (white cell count, ESR and c-reactive protein), serum lipoxin A4 and the cathelicidin LL-37, Forced Expired Volume in 1sec, Forced Vital Capacity and its ratio, transfer factor for carbon monoxide, antibiotic courses in 1 year, hospital admissions in 1 year and sputum microbiology. Phagocytosis and bacterial killing were assessed by both serum and airways neutrophils. From bronchoalveolar lavage fluid (BALF), I measured myeloperoxidase and neutrophil elastase. For both serum and BALF, lipidomics were obtained. (IV) To address the impact of anatomic compartment, gene expression was measured in from endobronchial brushings from the same cohort of bronchiectasis patients and controls as above, where samples were available. qPCR was performed for the following eicosanoid biosynthetic genes- 5 Lipoxygenase (LOX), 15 LO-A, 15LO-B and leukotriene (LT) A4 hydrolase. (V) To assess the anti inflammatory and pro resolution effect of LXA4 on neutrophils and monocyte derived macrophages from healthy volunteers, freshly isolated PMN will be treated with LXA4 or vehicle control. Spontaneous apoptosis was measured. fMLF and cytochalasin B was added and the inflammatory response assessed measuring myeloperoxidase (MPO), free neutrophil elastase (NE), CD11b, CD18 and CD62L. Human monocytes and PMNs were isolated from bronchiectasis patients. Following differentiation, LXA4 treated or control adherent, washed MDMs will be incubated with apoptotic stained PMNs. Efferocytosis was analyzed by flow cytometry. (VI) To establish the effect of Lipoxin A4 on neutrophil function following antibiotic treatment, the same study group used to evaluate aim 1 was taken. As a control group, 6 community acquired pneumonia patients at the beginning (day1) and the end (day 5) of oral or intravenous antibiotic therapy for infection were studied. Induced sputum and peripheral blood was taken at day1 and 5, where able. Phagocytosis and killing of GFP labeled bacteria and the effect of Lipoxin A4 was assessed and the two groups compared. Serum and sputum lipidomics were obtained in bronchiectasis exacerbations on day 1 and day 14. Serum lipidomics was obtained in pneumonia on day 1 and day 5. RESULTS (I) Neutrophil sub type study (Studied on healthy volunteers/ mild/ severe bronchiectasis) Peripheral blood neutrophils from bronchiectasis patients showed that there was significantly more viable neutrophils in mild and severe bronchiectasis compared to healthy volunteers, p=0.002 and p=0.005 respectively. In addition, there was significantly less apoptotic neutrophils in mild and severe bronchiectasis compared to healthy volunteers, p=0.0003 and p<0.0001 respectively. There was a significantly higher level of CD11b in the mild (p=0.01) and severe bronchiectasis (p=0.01) compared to healthy volunteers. There was more CD62L shedding (p=0.02) and myeloperoxidase release (p=0.04) in bronchiectasis compared to healthy volunteers. There was lesser phagocytosis in mild (p=0.04) and severe (p=0.03) bronchiectasis compared to healthy volunteers. This led to lesser bacterial killing in mild (p=0.04) and severe (p=0.0004) bronchiectasis compared to healthy volunteers. On comparison of serum to airways neutrophils, peripheral blood neutrophils had higher phagocytic capacity (p<0.0001 for both mild and severe) and bacterial killing (p=0.02 for both mild and severe) than airways neutrophils. During exacerbations, there was significantly higher phagocytosis (p=0.02) and killing at the end of exacerbation (p=0.03). Similarly, in community-acquired pneumonia, there was significantly higher phagocytosis (p=0.03) and killing at the end of exacerbation (p=0.03). When I compared bronchiectasis exacerbations with pneumonia, I found although there was no difference in phagocytosis, there was significantly higher bacterial killing in pneumonia than bronchiectasis, both at the start (p<0.0001) and at the end (p<0.0001) of infection. (II) Longitudinal cohort study in bronchiectasis In patients sufficient in serum LXA4 (>135 pg/ml) they had lesser antibiotic courses for an exacerbation (p=0.02), lesser levels of LTB4 (p<0.0001) and myeloperoxidase (p<0.0001), over the course of a year compared to lipoxin deficient patients. (III) Bronchoscopy study (Studied on healthy volunteers/ mild/ severe bronchiectasis) In serum, there was significantly higher levels of Lipoxin A4 detected in healthy volunteers compared to bronchiectasis patients, p=0.04. In peripheral blood neutrophils, there was a statistically significant improvement in phagocytosis by LXA4 in mild, moderate and severe bronchiectasis groups, p=0.01, p<0.0001 and p=0.01 respectively. Additionally, there was a statistically significant improvement in bacterial killing by LXA4 in mild, moderate and severe bronchiectasis groups, p=0.04, p=0.03 and p=0.01 respectively, in a dose dependent manner. In airways neutrophils, in the unaffected segments, there was no significant improvement in phagocytosis with LXA4 100nM in any of the groups. In the affected segments, there was a statistically significant improvement in phagocytosis by LXA4 100nM in the mild, moderate and severe bronchiectasis groups by LXA4 100nM, p=0.01, p=0.02 and p=0.04 respectively. In the unaffected segments, there was a statistically significant improvement in bacterial killing with LXA4 100nM in the severe group of patients only; p=0.02. In the affected segments, there was a statistically significant improvement in killing by LXA4 in the mild, moderate and severe bronchiectasis groups by LXA4 100nM, p=0.02, p=0.0005 and p=0.04 respectively. There were significantly higher levels of myeloperoxidase (p=0.002) and neutrophil elastase (p=0.005) detected in bronchoalveolar lavage fluid from severe patients. Serum lipidomics showed that there were significantly higher levels of PGE2, 15 HETE and LTB4 in patients with moderate-severe disease compared to healthy controls, p=0.03, p=0.03 and p=0.02 respectively. BALF lipidomics showed that there were significantly higher levels of PGE2, 5HETE and 15 HETE in patients with moderate-severe disease compared to patients with mild disease and healthy volunteers, p<0.0001, p=0.004 and p=0.005 respectively. There were significantly higher levels of 9HODE and LTB4 in moderate-severe patients compared to mild patients and healthy volunteers, p=0.04 and p<0.0001 respectively. (IV) Gene expression (Studied on mild and moderate- severe bronchiectasis) The delta Ct values for 5-LOX (p=0.01) and 15-LO-B (p=0.01) were both significantly increased in subjects with moderate-severe bronchiectasis compared with subjects with mild bronchiectasis. Delta Ct value of LTA4 hydrolase was significantly decreased in moderate-severe bronchiectasis compared to mild bronchiectasis (p=0.007), indicating increased expression of in moderate-severe bronchiectasis. LTA4 hydrolase was the most abundant RNA (lowest DCt) in bronchial brushings. (V) In vitro studies of lipoxin on neutrophil function (Studied on healthy volunteers/ mild/ severe bronchiectasis) Lipoxin was unable to modulate spontaneous apoptosis in any of the three groups; p=0.4, p=0.5 and p=0.4 in healthy, mild and severe bronchiectasis, respectively. There was a significant reduction in upregulation of CD11b by LXA4 in a dose dependent manner in healthy volunteers, mild bronchiectasis and severe bronchiectasis patients; p= 0.005, p=0.008 and p=0.01 respectively. There was a significant improvement in shedding of CD62L by LXA4 in a dose dependent manner in healthy volunteers, mild bronchiectasis and severe bronchiectasis patients; p=0.01, p=0.03 and p=0.04 respectively. There was a significant reduction in myeloperoxidase release by LXA4 in healthy volunteers, mild bronchiectasis and severe bronchiectasis patients; p= 0.02, p=0.04 and p=0.02 respectively. There was significant reduction of superoxide release by LXA4 in healthy volunteers (p=0.004) and severe bronchiectasis (p=0.03) but not on mild bronchiectasis (p=0.1). Lastly, on assessing bacterial phagocytosis and killing, there was a significant increase in phagocytosis pre treatment with LXA4, led to a dose dependent increase in phagocytosis in healthy volunteers (p=0.0001), mild bronchiectasis (p<0.0001) and severe bronchiectasis (p=0.03). Similarly, Lipoxin A4 led to a dose dependent increase in killing of GFP PAO1 in healthy volunteers (p=0.001), mild bronchiectasis (p=0.04) and severe bronchiectasis (p=0.01). (VI) Exacerbations In peripheral blood neutrophils, LXA4 was able to significantly improve bacterial phagocytosis (start p=0.03, end p=0.004) and killing (start p=0.01, end p=0.007) (blood neutrophils) both at the start and end of pneumonia exacerbations. In airways neutrophils, LXA4 was did not improve phagocytosis but improved bacterial killing at the start (p=0.02) and end (p=0.01) of exacerbation in bronchiectasis. In community acquired pneumonia, LXA4 was able to significantly improve bacterial phagocytosis (start p=0.01, end p=0.03) and killing (start p=0.04, end p=0.01) both at the start and end of pneumonia. CONCLUSION (i) Peripheral blood neutrophils are reprogrammed and persist longer in bronchiectasis. These reprogrammed neutrophils are the key contributors to the ongoing persistence of inflammation in bronchiectasis. (ii) There is a dysregulation of lipid mediators in the serum as well as in the airways of bronchiectasis patients. Lipoxin is able to override the pro survival signals in these reprogrammed neutrophils, stabilize and improve neutrophil function

Dysregulation of Prostaglandins, Leukotrienes and Lipoxin A4 in Bronchiectasis

Introduction: Bronchiectasis is characterised by excessive neutrophilic inflammation. Lipid mediators such as prostaglandins and leukotrienes have crucial roles in the inflammatory response. Further characterisation of these lipids and understanding the interplay of anti-inflammatory and proinflammatory lipid mediators could lead to the development of novel anti-inflammatory therapies for bronchiectasis. Aim: The aim of our study was to characterise the lipids obtained from serum and airways in patients with bronchiectasis in the stable state. Methods: Six healthy volunteers, 10 patients with mild bronchiectasis, 15 with moderate bronchiectasis and 9 with severe bronchiectasis were recruited. All participants had 60 mL of blood taken and underwent a bronchoscopy while in the stable state. Lipidomics was done on serum and bronchoalveolar lavage fluid (BALF). Results: In the stable state, in serum there were significantly higher levels of prostaglandin E2 (PGE2), 15-hydroxyeicosatetranoic acid (15-HETE) and leukotriene B4 (LTB4) in patients with moderate–severe disease compared with healthy volunteers. There was a significantly lower level of lipoxin A4 (LXA4) in severe bronchiectasis. In BALF, there were significantly higher levels of PGE2, 5-HETE, 15-HETE, 9-hydroxyoctadecadienoic acid and LTB4 in moderate–severe patients compared with healthy volunteers. In the stable state, there was a negative correlation of PGE2 and LTB4 with % predicted forced expiratory volume in 1 s and a positive correlation with antibiotic courses. LXA4 improved blood and airway neutrophil phagocytosis and bacterial killing in patients with bronchiectasis. Additionally LXA4 reduced neutrophil activation and degranulation. Conclusion: There is a dysregulation of lipid mediators in bronchiectasis with excess proinflammatory lipids. LXA4 improves the function of reprogrammed neutrophils. The therapeutic efficacy of LXA4 in bronchiectasis warrants further studies

The impact of acute air pollution fluctuations on bronchiectasis pulmonary exacerbation:A case-crossover analysis

In bronchiectasis, exacerbations are believed to be triggered by infectious agents, but often no pathogen can be identified. We hypothesised that acute air pollution exposure may be associated with bronchiectasis exacerbations.We combined a case-crossover design with distributed lag models in an observational record linkage study. Patients were recruited from a specialist bronchiectasis clinic at Ninewells Hospital, Dundee, UK.We recruited 432 patients with clinically confirmed bronchiectasis, as diagnosed by high-resolution computed tomography. After excluding days with missing air pollution data, the final model for particles with a 50% cut-off aerodynamic diameter of 10 µm (PM; 10; ) was based on 6741 exacerbations from 430 patients and for nitrogen dioxide (NO; 2; ) it included 6248 exacerbations from 426 patients. For each 10 µg·m; -; ³ increase in PM; 10; and NO; 2; , the risk of having an exacerbation that same day increased significantly by 4.5% (95% CI 0.9-8.3) and 3.2% (95% CI 0.7-5.8) respectively. The overall (lag zero to four) increase in risk of exacerbation for a 10 μg·m; -3; increase in air pollutant concentration was 11.2% (95% CI 6.0-16.8) for PM; 10; and 4.7% (95% CI 0.1-9.5) for NO; 2; Subanalysis showed higher relative risks during spring (PM; 10; 1.198 (95% CI 1.102-1.303), NO; 2; 1.146 (95% CI 1.035-1.268)) and summer (PM; 10; 2.142 (95% CI 1.785-2.570), NO; 2; 1.352 (95% CI 1.140-1.602)) when outdoor air pollution exposure would be expected to be highest.In conclusion, acute air pollution fluctuations are associated with increased exacerbation risk in bronchiectasis

The BRICS (Bronchiectasis Radiologically Indexed CT Score)- a multi-center study score for use in idiopathic and post infective bronchiectasis

OBJECTIVES: The goal of this study was to develop a simplified radiological score that could assess clinical disease severity in bronchiectasis. METHODS: The Bronchiectasis Radiologically Indexed CT Score (BRICS) was devised based on a multivariable analysis of the Bhalla score and its ability in predicting clinical parameters of severity. The score was then externally validated in six centers in 302 patients. RESULTS: A total of 184 high-resolution CT scans were scored for the validation cohort. In a multiple logistic regression model, disease severity markers significantly associated with the Bhalla score were percent predicted FEV1, sputum purulence, and exacerbations requiring hospital admission. Components of the Bhalla score that were significantly associated with the disease severity markers were bronchial dilatation and number of bronchopulmonary segments with emphysema. The BRICS was developed with these two parameters. The receiver operating-characteristic curve values for BRICS in the derivation cohort were 0.79 for percent predicted FEV1, 0.71 for sputum purulence, and 0.75 for hospital admissions per year; these values were 0.81, 0.70, and 0.70, respectively, in the validation cohort. Sputum free neutrophil elastase activity was significantly elevated in the group with emphysema on CT imaging. CONCLUSIONS: A simplified CT scoring system can be used as an adjunct to clinical parameters to predict disease severity in patients with idiopathic and postinfective bronchiectasis

An investigation into the controllability of multivalued stochastic fractional differential inclusions

This research aims to investigate the approximate controllability of multivalued impulsive stochastic fractional differential inclusions in Hilbert space with ABC fractional-order derivatives. First, we confirm the existence of mild solutions for the proposed control system using stochastic analysis, resolvent operator theory, and the fixed point technique. Secondly, we discuss a new set of sufficient conditions for the approximate controllability of the systems. The results are obtained under the assumption that the associated linear system is approximately controllable. Finally, an example is provided to illustrate the obtained results