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.
- neutrophil biology
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/licenses/by/4.0/.
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What is the key question?
Is there a dysregulation of lipids in bronchiectasis that leads to unremitting and chronic inflammation?
What is the bottom line?
Ex vivo, lipoxin A4 can improve the function of reprogrammed blood and airway neutrophils in bronchiectasis.
Why read on?
This is the first study assessing the role of lipids in bronchiectasis in depth. Here we have demonstrated that dysregulation of the proinflammatory and anti-inflammatory lipids in bronchiectasis contribute to failure of resolution of inflammation. Targeting the lipid pathways to initiate the resolution process in bronchiectasis may lead to development of novel non-antibiotic therapy in the stable state.
Excessive inflammation is widely accepted to be a unifying component in many chronic diseases, including bronchiectasis, vascular diseases, metabolic syndrome and neurological diseases, and thus is a public health concern. Understanding endogenous control points within the inflammatory response could potentially provide us with new perspectives on disease pathogenesis and treatment approaches. Break in the barrier, trauma and microbial invasion encourage the host to clear microbial pathogens, remodel and regenerate tissue. The acute inflammatory response is protective and usually self-limiting. Oxidative lipid products including, notably, the products of unsaturated lipids are increasingly being recognised as important contributors to chronic inflammatory diseases.1 2 Eicosanoids3 4 produced from the n-6 polyunsaturated fatty acid, arachidonic acid (20:4), as well as many cytokines and chemokines,5 6 have crucial roles in the initial response. Interactions among prostaglandins, leukotrienes and proinflammatory cytokines amplify inflammation, the signs and effects of which can be reduced by pharmacological inhibition and receptor antagonists.3–5 However, given that excessive inflammation contributes to several widely occurring diseases, improvements are required in treatment and in our understanding of the mechanisms involved. Eicosanoids also include lipoxins that possess potent anti-inflammatory properties.7 Further, n-3 fatty acids (eicosapentaenoic acid 20:5, and docosahexaenoic acid, 22:6) are precursors for proresolving mediators such as resolvins and maresins that limit the duration and magnitude of inflammatory responses.8 Recent advances have facilitated more detailed profiling of lipid mediators in serum and at the sites of inflammation.9–11 Characterisation of these lipids could thereby lead to the development of novel anti-inflammatory therapies for chronic inflammatory conditions.
Bronchiectasis is characterised by recurrent cough, daily sputum production and recurrent chest infections. There is excessive neutrophilic inflammation, but the driver for this unremitting inflammation is not known. We hypothesise that there is a failure of resolution of inflammation in bronchiectasis. Recently, we have been investigating the lipid pathway to establish if there is a dysregulation of the lipids in bronchiectasis contributing to the chronic inflammation. While this could be contributing to the persistent chronic inflammatory state in bronchiectasis, there is currently no data in the literature to indicate the interplay of lipid mediators in bronchiectasis.
The aim of our study was to characterise the lipids obtained from blood and airway samples in patients with bronchiectasis in the stable state and to assess the efficacy of lipoxin A4 (LXA4) on neutrophil function.
Six healthy volunteers (partners of the patients recruited in the study with no background medical conditions and not currently on medication), 10 patients with mild bronchiectasis, 15 with moderate and 9 with severe bronchiectasis were recruited. All participants had 60 mL of blood taken and underwent a bronchoscopy. Two segments of the lungs were washed out in patients with bronchiectasis, an area affected by bronchiectasis and an area unaffected by bronchiectasis, predetermined by CT scan of chest. This led to patients acting as their own internal control. Lipidomics was done on blood and bronchoalveolar samples were obtained.
The severity of bronchiectasis was calculated using the Bronchiectasis Severity Index (BSI).12 The BSI is a risk stratification tool for morbidity and mortality in bronchiectasis. The minimum score is 0 and the maximum score is 26. A score between 0 and 4 indicates mild disease; 5–8 indicates moderate disease; and a score of ≥9 indicate severe disease. The BSI was calculated in all patients with bronchiectasis taking part in the study.
Inclusion criteria include idiopathic or postinfective bronchiectasis, age >18 years and no infective exacerbation of bronchiectasis for at least 4 weeks prior to giving serum/bronchoscopy.
Patients on statin, aspirin, inhaled corticosteroids and long-term macrolides were excluded.
All participants underwent a bronchoscopy. Participants were sedated with midazolam±fentanyl. Bronchoalveolar lavage (BAL) and brushings were obtained. For patients with bronchiectasis, BAL was done in an area affected by bronchiectasis and in an area unaffected by bronchiectasis as identified on CT scans done prior to bronchoscopy.
Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of eicosanoids
Quantification of eicosanoids and related lipid mediators in patient bronchoalveolar lavage fluid (BALF) and serum samples was performed using LC-MS/MS13 (see online supplemental file, online supplemental figure 1 and online supplemental table T1).
LXA4 detection by ELISA
Serum LXA4 was measured as per manufacturer’s instruction (Neogen).
LXA4 was the lipid of choice for all the experiments outlined in this article as pilot data (data not shown in this paper) using sputum and blood from bronchiectasis exacerbations showed that LXA4 was the only lipid that improved after treatment with antibiotics.
Isolation of blood and airway neutrophils
Freshly drawn blood was collected into 3.8% sodium citrate, and granulocytes were subsequently isolated by dextran sedimentation and discontinuous Percoll gradient.14 Sputum and BAL were washed and treated with sputolysin, and airway neutrophils were isolated. Anti-CD16 antibodies (Abcam) were used to identify neutrophils by flow cytometry.
LXA4 function on reprogrammed bronchiectasis neutrophils
We assessed phagocytosis and killing of green fluorescent protein (GFP)–Pseudomonas O1 (PAO1), spontaneous neutrophil apoptosis, neutrophil activation (CD62L/CD11b expression) and neutrophil degranulation (myeloperoxidase release). Further information on the specific experiments is provided in the online supplemental file.
Flow cytometry analysis was performed using FlowJo V.10.0.4 (Tree Star, Ashland, Oregon, USA). Results are presented as mean±SEM. Paired and unpaired t-tests were used to compare the two groups, where applicable. Data were analysed by one-way analysis of variance (ANOVA) with Bonferonni’s multiple comparison post hoc test (GraphPad Prism V.6; GraphPad Software, La Jolla, California, USA), when three groups were involved. A repeated measures ANOVA was used where samples from the same participant receive multiple treatments; significance was accepted with p values: *p<0.05.
Baseline demographics of the participants are shown in table 1.
LC-MS/MS was done on all serum samples obtained, where available. Only data that passed quality control were used, and thus we have a smaller sample size. The demographics are shown in online supplemental table 1. Samples were divided into mild9 and moderate–severe15 bronchiectasis groups (moderate and severe groups were combined as lipids were not detected in all samples by lipidomics, and hence data were combined to obtain meaningful interpretation. Baseline demographics of mild9 and moderate–severe15 groups are provided in online supplemental table T2.
The main lipids assessed were LXA4, resolvins, maresins, prostaglandin E2 (PGE2), 5-hydroxyeicosatetranoic acid (5-HETE), 15-hydroxyeicosatetranoic acid (15-HETE), leukotriene B4 (LTB4) and 9-hydroxyoctadecadienoic acid (9-HODE). 15-HETE is a precursor of LXA4 and is a proinflammatory mediator. LTB4 is a proinflammatory cytokine and 9-HODE is a proinflammatory metabolite produced from arachidonic acid.
There were significantly higher levels of PGE2, 15-HETE and LTB4 in patients with moderate–severe bronchiectasis compared with healthy controls (p=0.03, p=0.03 and p=0.02, respectively). Although there was a trend towards higher 5-HETE and 9-HODE levels in moderate–severe bronchiectasis compared with healthy volunteers, this failed to reach statistical significance (p=0.3 and 0.2, respectively). Although there was a trend towards higher PGE2, 15-HETE, LTB4, 5-HETE and 9-HODE levels between the mild bronchiectasis and healthy volunteers, this was not statistically significant (figure 1). LXA4, resolvins and maresins were not detected in any of the samples by LC-MS/MS.
Blood LXA4 measured by ELISA
LXA4 was detected by ELISA. Using one-way ANOVA, we found that there was a significantly lower level of LXA4 in severe bronchiectasis, p=0.04 (figure 2), as measured by ELISA.
There were significantly higher levels of PGE2, 5-HETE, 15-HETE, 9-HODE and LTB4 in patients with moderate–severe bronchiectasis compared with healthy volunteers (p<0.0001, p=0.004, p=0.005, p=0.04 and p<0.0001, respectively) (figure 3). Although there was a trend, there was no statistically significant difference in the PGE2, 15-HETE, LTB4, 5-HETE and 9-HODE levels between mild bronchiectasis and healthy volunteers. LXA4, resolvins and maresins were not detected in any of the samples by LC-MS/MS. PGE2 was not detectable in BALF from healthy individuals.
Correlation of blood PGE2 and LTB4 to markers of disease severity
There was a positive correlation of PGE2 with antibiotic courses (r=0.78, 95% CI 0.61 to 0.88, p<0.0001) and a negative correlation with % predicted forced expiratory volume in 1 s (FEV1) (r=−0.46, 95% CI −0.15 to −0.69, p=0.004). Similarly, there was a positive correlation of LTB4 with antibiotic courses (r=0.76, 95% CI 0.56 to 0.87, p<0.0001) and a negative correlation with % predicted FEV1 (r=−0.58, 95% CI −0.3 to −0.76, p=0.0003; figure 4). There was no correlation of PGE2 or LTB4 to other parameters of the disease severity on the BSI. There was no correlation of 15-HETE, 5-HETE and 9-HODE to markers of disease severity.
Effect of biological sex on PGE2
There was biological sex imbalance in our cohort, and to investigate this further, there was a subanalysis of blood PGE2. The blood PGE2 was 1.1 (±0.3) for male healthy volunteers and 1.3 (±0.5) for female healthy volunteers (p=0.2), 4.2 (±1.4) for male mild bronchiectasis and 3.9 (±0.1) for female mild bronchiectasis (p=0.8), 2.9 (±0.6) for male moderate–severe bronchiectasis, and 4.2 (±0.7) for female moderate–severe bronchiectasis (p=0.2). This is a small study and so detailed further subanalysis was not carried out.
Effect of LXA4 on blood neutrophil function
As we detected significantly lower levels of LXA4 in blood of patients with severe bronchiectasis, we investigated the effect of LXA4 on reprogrammed neutrophil function.15 With the complexity of the experiments, the authors studied healthy controls and patients with mild and severe bronchiectasis only. Blood neutrophils from healthy volunteers (n=6) and patients with mild (n=10) and severe bronchiectasis (n=9) were pretreated ex vivo with varying concentrations of LXA4 for 30 min and then coincubated with GFP–PAO1. Phagocytosis of bacteria (GFP–PAO1) was assessed after 15 min and killing after 24 hours. Total phagocytosis was calculated by assessing neutrophils positive for GFP. Data were analysed by gating the overall phagocytosis first. There was no difference in overall phagocytosis in healthy controls and in patients with mild and severe bronchiectasis. Next, we gated the neutrophils that had taken up much higher number of bacteria, as indicated by the mean fluorescence (fluorescence intensity, FLI). This was done at 50% of the total phagocytosis and we called this high MFLI phagocytosis. In healthy controls, mild and severe bronchiectasis, LXA4 was able to significantly improve phagocytosis (comparing only the high MFLI and not total phagocytosis) (figure 5A) and killing of GFP–PAO1 in a concentration-dependent manner with statistical significance achieved at 100 nM LXA4 (figure 5B).
Effect of LXA4 on airway neutrophil function
Airway neutrophils were isolated from BALF from patients with bronchiectasis (no airway neutrophils could be isolated from healthy volunteers), and phagocytosis and killing assays were performed using GFP–PAO1.
Neutrophils collected from affected lung segments demonstrated statistically significant improvement in phagocytosis (neutrophil gating done as in previous experiment) and killing after treatment with 100 nM LXA4 in mild, moderate and severe bronchiectases (figure 6A,B). The neutrophils collected from unaffected lung segments displayed significant improvement in bacterial killing after treatment with 100 nM LXA4 in patients with severe bronchiectasis only (p=0.02).
Effect of LXA4 on stable disease-state peripheral blood neutrophil spontaneous apoptosis, surface expression of CD11b and CD62L, and myeloperoxidase release
For this experiment, blood neutrophils from healthy volunteers and patients with stable-state bronchiectasis with either mild and moderate–severe disease were pretreated with LXA4 1, 10 and 100 nM and assessed for the onset of spontaneous apoptosis. LXA4 did not significantly modulate spontaneous apoptosis or viability of neutrophils (p=0.4, p=0.5 and p=0.4, respectively), in contrast to roscovitine, used as a positive control for apoptosis induction (online supplemental figure S2).
To evaluate the effect of LXA4 on fMLF-induced neutrophil activation, surface expression of CD11b and CD62L was measured. In this experiment, LXA4 treatment induced a small but statistically significant reduction in fMLF-induced upregulation of CD11b and shedding of CD62L in a concentration-dependent manner, in healthy volunteers and in patients with mild and moderate–severe bronchiectases (online supplemental figure S3 and 4).
Additionally, LXA4 reduced neutrophil degranulation and release of myeloperoxidase in a concentration-dependent manner from neutrophils isolated from healthy volunteers and in patients with mild and moderate–severe bronchiectasis (online supplemental figure S5).
This is the first study characterising the lipid profile in bronchiectasis blood and airways in the stable state. We established that there is a dysregulation of the lipids in serum and airways in the stable state. In serum, there was significantly higher levels of the proinflammatory metabolites PGE2, 15-HETE and LTB4 and significantly lower levels of the anti-inflammatory mediator LXA4 in severe disease compared with mild disease and healthy volunteers. Although LXA4 was not detectable in serum using LC-MS/MS, it was detected by ELISA. This can be explained by level of sensitivity of the assays used. Detection limits were in nanogram per millilitre by LC-MS and in picogram per millilitre by ELISA. In the airways, there were significantly higher levels of PGE2, 5-HETE, 15-HETE, 9-HODE and LTB4. There was a correlation of PGE2 and LTB4 with antibiotic courses and % predicted FEV1. Higher levels of PGE2 and LTB4 were inversely related to the % predicted FEV1 and directly related to the number of antibiotic courses for bronchiectasis exacerbations.
In the stable state, LXA4 was able to significantly improve phagocytosis and killing of GFP Pseudomonas aeruginosa by blood and airway neutrophils, in a concentration-dependent manner. In addition, the authors demonstrated that LXA4 reduced fMLF-induced neutrophil activation. However, there was no effect of LXA4 on spontaneous neutrophil apoptosis.
To the authors’ best knowledge, prostaglandins have not been studied in bronchiectasis. In COPD, PGE2 levels are increased in the exhaled breath and are known to correlate with airflow obstruction.16 17 Additionally, studies have demonstrated that PGE2 is a critical component in amplifying and perpetuating senescence and inflammation in COPD fibroblasts.18 PGE2 enhances LTB4-mediated polymorphonuclear leucocyte extravasation and tissue injury that is blocked by topical administration of synthetic LXA4.19 However, prostaglandins are also key in the temporal switch of LTB4 to LXA4—a term coined as ‘lipid mediator class switching’.20 In the stable state, the authors demonstrated that PGE2 and LTB4 were significantly higher and LXA4 was significantly lower in blood in severe bronchiectasis. Serum PGE2 and LTB4 levels were correlated to airflow obstruction as measured by FEV1. Higher serum PGE2 and LTB4 levels were also correlated to more exacerbations requiring antibiotic courses in bronchiectasis, both markers of disease severity in bronchiectasis.12 Studies have established that disease severity in bronchiectasis predicts mortality, hospital admissions, exacerbations, quality of life, respiratory symptoms, exercise capacity and lung function decline in bronchiectasis.21 This dysregulation between lipid mediators that the authors have shown here would thereby lead to more inflammation even in the stable state. Although PGE2 levels are known to initiate the class switching during resolution of inflammation, this was not demonstrated in our study. Almost certainly, the levels of LXA4 detected in bronchiectasis serum are unable to counter-regulate the production of the proinflammatory LTB4.
In addition to PGE2 and LTB4, the other metabolites (5-HETE, 15-HETE and 9-HODE) detected in our study are also proinflammatory mediators, and they remain elevated even in the stable state in moderate and severe bronchiectases compared with mild disease. LTB4 has been studied in bronchiectasis and is known to be one of the major chemotactic factors in the bronchial airways in bronchiectasis.22 Additionally, there is evidence to suggest that LTB4 is raised during an exacerbation and reduces with antibiotic therapy.23 Persistently elevated lipid mediators in bronchiectasis, in part, may explain why more patients with moderate–severe bronchiectasis have a higher mortality rate and more hospital admissions compared with patients with mild disease.12
LXA4 was able to improve blood and airway neutrophil bacterial phagocytosis and killing, thereby enhancing bacterial clearance and potentially having long-term consequences on infection, inflammation and resolution. The impact of LXA4 on bronchiectasis airway neutrophil function was more pronounced when neutrophils were isolated from a disease-affected lung region than an unaffected region. The mechanism underpinning this observation remains to be determined, but this raises the intriguing possibility that regional lung patterning of LXA4 deficiency might exist, with cells collected having had divergent in vivo exposure to LXA4 before experimental use.
LXA4 was able to reduce fMLF-induced CD11b upregulation, CD62L shedding and myeloperoxidase release. These anti-inflammatory functions of LXA4 have previously been demonstrated in the literature.17 However, this is the first time that the effects of lipoxins have been demonstrated on a subset of reprogrammed neutrophils from patients with bronchiectasis.
Biological sex may have an effect on the dynamics of the lipids in bronchiectatic airways. We did not identify differences with blood PGE2, but as this was a small study, further detailed subanalysis was not carried out. Further studies would be needed to explore this.
The authors have shown that serum neutrophils are reprogrammed in bronchiectasis, leading to persistent and unresolving inflammation.15 This study now demonstrates that there is a dysregulation of the lipid mediators and failure of class switching during inflammation in bronchiectasis, despite adequate levels of PGE2. Whether there is a role of cyclo-oxygenase inhibitors in bronchiectasis to block the production of PGE2 needs to be explored further. Certainly, with the emergence of antibiotic resistance, the role of novel specialised proresolving lipid mediators is promising, especially in bronchiectasis, where recurrent exacerbations requiring antibiotic therapy is one of the cardinal features of the disease.
Limitations of the study
There are a couple of limitations in this study: first, that this is a small study, and second, this study did not assess the role LXA4 on bronchiectasis neutrophils during exacerbations.
There is a dysregulation of lipid mediators in bronchiectasis in the stable state with excess proinflammatory lipids. LXA4 improves the function of reprogrammed neutrophils. The therapeutic efficacy of LXA4 in bronchiectasis warrants further studies.
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
Patient consent for publication
Lothian Research Ethics Committee gave approval for the stud (10/S1402 /S1402/33).
The authors thank T Tolker Nielsen, University of Copenhagen, for providing the GFP Pseudomonas aeruginosa, and the QMRI Flow Cytometry and Cell Sorting Facility for assistance with flow cytometric analysis.
Contributors PB performed the experiments, collected and interpreted the data and wrote the manuscript. KZ performed lipidomics and contributed to the writing of the manuscript. PDW contributed to interpretation of data and writing of the manuscript. DJD, AGR and ATH contributed to experimental design, interpretation of data and writing of the manuscript.
Funding PB was funded by Chief Scientist Office for this study (CAF/13/02). KZ and PDW gratefully acknowledge the financial support of the European Regional. DJD was supported by a Medical Research Council Senior Non-clinical Fellowship (G1002046). AGR was supported by Medical Research Council (grant MR/K013386/1) Development Fund, Scottish Funding Council and Highlands and Islands Enterprise.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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