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