Assessing the Anti-Inflammatory Effects of an Orally Dosed Enzymatically Liberated Fish Oil in a House Dust Model of Allergic Asthma.

Eosinophils are a major driver of inflammation in a number of human diseases, including asthma. Biologic therapies targeting IL-5 have enabled better control of severe eosinophilic asthma, but no such advances have been made for enhancing the control of moderate asthma. However, a number of moderate asthma sufferers remain troubled by unresolved symptoms, treatment side effects, or both. OmeGo, an enzymatically liberated fish oil, has demonstrated antioxidant and anti-inflammatory properties including the reduction of eosinophilia. A house dust mite model of induced asthma in mice was utilized in this study, and OmeGo showed a significant reduction in eosinophilic lung and systemic inflammation and reduced lung remodelling compared to cod liver oil. The CRTH2 antagonist fevipiprant showed an anti-inflammatory profile similar to that of OmeGo. OmeGo has the potential to be a pragmatic, cost-effective co-treatment for less severe forms of eosinophilic asthma. Proof-of-concept studies are planned.

Differential responses of COPD macrophages to respiratory bacterial pathogens.

COPD patients have increased susceptibility to airway bacterial colonisation. Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae are three of the most common respiratory bacterial species in COPD. H. influenzae colonisation, but not other bacteria, in COPD patients is associated with higher sputum neutrophil counts. Alveolar macrophages are key in clearance of bacteria as well as releasing mediators to recruit and activate other immune cells in response to infection. The aim was to characterise differences in COPD macrophage responses to H. influenzae, M. catarrhalis and S. pneumoniae, focusing on release of inflammatory and chemotactic mediators, and apoptosis regulation. Lung macrophages and monocyte-derived macrophages from COPD patients and control subjects were exposed to H. influenzae, M. catarrhalis or S. pneumoniae. Cytokine secretion (tumour necrosis factor-α, interleukin (IL)-6, CXCL8, CCL5 and IL-1ß) were measured by ELISA and quantitative reverse transcriptase PCR (RT-qPCR), and apoptosis genes MCL-1, BCL-2, BAX and BAK1 by RT-qPCR. Apoptosis and reactive oxygen species (ROS) release were also measured. Macrophages responded differentially to the bacterial species, with increased, prolonged production of the neutrophil chemoattractant CXCL8 in response to H. influenzae and M. catarrhalis but not S. pneumoniae. S. pneumoniae initiated macrophage apoptosis and ROS release, H. influenzae and M. catarrhalis did not and increased anti-apoptosis gene expression (BCL-2 5.5-fold and MCL-1 2.4-fold, respectively). Differential cytokine responses of macrophages to these bacterial species can explain neutrophilic airway inflammation associated with H. influenzae, but not S. pneumoniae in COPD. Furthermore, delayed macrophage apoptosis is a potential mechanism contributing to inability to clear H. influenzae.

Red Blood Cell-Derived Iron Alters Macrophage Function in COPD.

Lung macrophage iron levels are increased in COPD patients. Lung macrophage iron levels are thought to be increased by cigarette smoke, but the role of red blood cells (RBCs) as a source of iron has not been investigated. We investigate RBCs as a potential source of alveolar iron in COPD, and determine the effect of RBC-derived iron on macrophage function. We used lung tissue sections to assess RBC coverage of the alveolar space, iron and ferritin levels in 11 non-smokers (NS), 15 smokers (S) and 32 COPD patients. Lung macrophages were isolated from lung resections (n = 68) and treated with hemin or ferric ammonium citrate (50, 100 or 200 µM). Lung macrophage phenotype marker gene expression was measured by qPCR. The phagocytosis of Non-typeable Haemophilus influenzae (NTHi) was measured by flow cytometry. Cytokine production in response to NTHi in iron-treated macrophages was measured by ELISA. Lung macrophage iron levels were significantly correlated with RBC coverage of the alveolar space (r = 0.31, p = 0.02). Furthermore, RBC coverage and lung macrophage iron were significantly increased in COPD patients and correlated with airflow obstruction. Hemin treatment downregulated CD36, CD163, HLA-DR, CD38, TLR4, CD14 and MARCO gene expression. Hemin-treated macrophages also impaired production of pro-inflammatory cytokines in response to NTHi exposure, and decreased phagocytosis of NTHi (200 µM: 35% decrease; p = 0.03). RBCs are a plausible source of pulmonary iron overload in COPD. RBC-derived iron dysregulates macrophage phenotype and function.

Inhaled Phosphodiesterase Inhibitors for the Treatment of Chronic Obstructive Pulmonary Disease.

Phosphodiesterase (PDE) 4 inhibitors prevent the metabolism of cyclic adenosine monophosphate, thereby reducing inflammation. Inhaled PDE4 inhibitors aim to restrict systemic drug exposure to enhance the potential for clinical benefits (in the lungs) versus adverse events (systemically). The orally administered PDE4 inhibitor roflumilast reduces exacerbation rates in the subgroup of chronic obstructive pulmonary disease patients with a history of exacerbations and the presence of chronic bronchitis, but can cause PDE4 related adverse effects due to systemic exposure. CHF6001 is an inhaled PDE4 inhibitor, while inhaled ensifentrine is an inhibitor of both PDE3 and PDE4; antagonism of PDE3 facilitates smooth muscle relaxation and hence bronchodilation. These inhaled PDE inhibitors have both reported positive findings from early phase clinical trials, and have been well tolerated. Longer term trials are needed to firmly establish the clinical benefits of these drugs.

Airway Bacteria Quantification Using Polymerase Chain Reaction Combined with Neutrophil and Eosinophil Counts Identifies Distinct COPD Endotypes.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) inflammatory endotypes are associated with different airway microbiomes. We used quantitative polymerase chain reaction (qPCR) analysis of sputum samples to establish the bacterial load upper limit in healthy controls; these values determined the bacterial colonisation prevalence in a longitudinal COPD cohort. Bacteriology combined with sputum inflammatory cells counts were used to investigate COPD endotypes. METHODS: Sixty COPD patients and 15 healthy non-smoking controls were recruited. Sputum was analysed by qPCR (for Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and Psuedomonas aeruginosa) and sputum differential cell counts at baseline and 6 months. RESULTS: At baseline and 6 months, 23.1% and 25.6% of COPD patients were colonised with H. influenzae, while colonisation with other bacterial species was less common, e.g., S. pneumoniae-1.9% and 5.1%, respectively. H. influenzae + ve patients had higher neutrophil counts at baseline (90.1% vs. 67.3%, p < 0.01), with similar results at 6 months. COPD patients with sputum eosinophil counts ≥3% at ≥1 visit rarely showed bacterial colonisation. CONCLUSIONS: The prevalence of H. influenzae colonisation was approximately 25%, with low colonisation for other bacterial species. H. influenzae colonisation was associated with sputum neutrophilia, while eosinophilic inflammation and H. influenzae colonisation rarely coexisted.

Assessment of bacterial exposure on phagocytic capability and surface marker expression of sputum macrophages and neutrophils in COPD patients.

Defective phagocytosis has been shown in chronic obstructive pulmonary disease (COPD) bronchoalveolar lavage and blood monocyte-derived macrophages. Phagocytic capabilities of sputum macrophages and neutrophils in COPD are unknown. We investigated phagocytosis in these cells from COPD patients and controls. Phagocytosis of Streptococcus pneumoniae or fluorescently labelled non-typeable Haemophilus influenzae (NTHi) by sputum macrophages and neutrophils was determined by gentamycin protection assay (COPD; n = 5) or flow cytometry in 14 COPD patients, 8 healthy smokers (HS) and 9 healthy never-smokers (HNS). Sputum macrophages and neutrophils were differentiated by adherence for the gentamycin protection assay or receptor expression (CD206 and CD66b, respectively), by flow cytometry. The effects of NTHi on macrophage expression of CD206 and CD14 and neutrophil expression of CD16 were determined by flow cytometry. There was greater uptake of S. pneumoniae [~10-fold more colony-forming units (CFU)/ml] by sputum neutrophils compared to macrophages in COPD patients. Flow cytometry showed greater NTHi uptake by neutrophils compared to macrophages in COPD (67 versus 38%, respectively) and HS (61 versus 31%, respectively). NTHi uptake by macrophages was lower in HS (31%, p = 0.019) and COPD patients (38%, p = 0.069) compared to HNS (57%). NTHi uptake by neutrophils was similar between groups. NTHi exposure reduced CD206 and CD14 expression on macrophages and CD16 expression on neutrophils. Sputum neutrophils showed more phagocytic activity than macrophages. There was some evidence that bacterial phagocytosis was impaired in HS sputum macrophages, but no impairment of neutrophils was observed in HS or COPD patients. These results highlight the relative contributions of neutrophils and macrophages to bacterial clearance in COPD.

Arteriovenous Fistula Surveillance Using Tomographic 3D Ultrasound.

OBJECTIVE: A well functioning arteriovenous fistula (AVF) is essential for haemodialysis. Despite regular duplex ultrasound (DUS) a significant number of AVFs fail. Tomographic 3D ultrasound (tUS) creates a 3D image of the AVF that can be interpreted by the clinician. DUS, tUS, and fistulograms were compared for the identification and measurement of flow limiting stenosis. METHODS: Patients with AVF dysfunction on routine Transonic surveillance, defined as (1) > 15% reduction in flow on two consecutive occasions, (2) > 30% reduction in flow on one occasion, (3) flow of < 600 mL/sec, (4) presence of recirculation, underwent DUS. AVF tUS imaging was performed prior to fistulography. All fistulograms were reported by the same consultant radiologist and tUS images by the same vascular scientist blinded to the fistulogram results. Maximum diameter reduction in all stenoses were measured using all three imaging techniques. RESULTS: In 97 patients with 101 stenoses, the mean (± standard deviation [SD]) severity of stenosis was 63.0 ± 13.9%, 65.0 ± 11.6%, and 64.8 ± 11.7% for the fistulograms, DUS, and tUS respectively. The mean (± SD) time between ultrasound and fistulography imaging was 15.0 ± 14.5 days. Assuming the fistulogram as the "gold standard", Bland-Altman agreement for DUS was -1.9 ± 15.5% (limit of agreement [LOA] -32.2 - 28.4) compared with -1.7 ± 15.4% (LOA -31.9 - 28.4) for tUS. Median (± interquartile range) time to complete the investigation was 09:00 ± 03:19 minutes for DUS and 03:13 ± 01:56 minutes for tUS (p < .001). CONCLUSION: DUS and tUS were equally accurate at detecting AVF complications but tUS investigation requires less skill and was significantly quicker than DUS.

Dysregulation of the CD163-Haptoglobin Axis in the Airways of COPD Patients.

Pulmonary iron levels are increased in chronic obstructive pulmonary disease (COPD) patients. Iron causes oxidative stress and is a nutrient for pathogenic bacteria. Iron may therefore play an important role in the pathophysiology of COPD. The CD163-haptglobin axis plays a central role in the regulation of iron bioavailability. The aim of this study was to examine dysregulation of the CD163-haptglobin axis in COPD. We measured soluble CD163 (sCD163) and haptoglobin levels in sputum supernatants by enzyme-linked immunosorbent assay (ELISA) and sputum macrophage CD163 and haptoglobin expression by flow cytometry in COPD patients and controls. SCD163 levels were lower in COPD patients compared to controls (p = 0.02), with a significant correlation to forced expiratory volume in 1 s (FEV1)% predicted (rho = 0.5, p = 0.0007). Sputum macrophage CD163 expression was similar between COPD patients and controls. SCD163 levels and macrophage CD163 expression were lower in COPD current smokers compared to COPD ex-smokers. Haptoglobin levels were not altered in COPD patients but were regulated by genotype. Macrophage CD163 and haptolgobin expression were significantly correlated, supporting the role of CD163 in the cellular uptake of haptoglobin. Our data implicates a dysfunctional CD163-haptoglobin axis in COPD, which may contribute to disease pathophysiology, presumably due to reduced clearance of extracellular iron.

Effects of corticosteroids on COPD lung macrophage phenotype and function.

The numbers of macrophages are increased in the lungs of chronic obstructive pulmonary disease (COPD) patients. COPD lung macrophages have reduced ability to phagocytose microbes and efferocytose apoptotic cells. Inhaled corticosteroids (ICSs) are widely used anti-inflammatory drugs in COPD; however, their role beyond suppression of cytokine release has not been explored in COPD macrophages. We have examined the effects of corticosteroids on COPD lung macrophage phenotype and function. Lung macrophages from controls and COPD patients were treated with corticosteroids; effects on gene and protein expression of CD163, CD164, CD206, MERTK, CD64, CD80 and CD86 were studied. We also examined the effect of corticosteroids on the function of CD163, MERTK and cluster of differentiation 64 (CD64). Corticosteroid increased CD163, CD164, CD206 and MERTK expression and reduced CD64, CD80 and CD86 expression. We also observed an increase in the uptake of the haemoglobin-haptoglobin complex (CD163) from 59 up to 81% and an increase in efferocytosis of apoptotic neutrophils (MERTK) from 15 up to 28% following corticosteroid treatment. We observed no effect on bacterial phagocytosis. Corticosteroids alter the phenotype and function of COPD lung macrophages. Our findings suggest mechanisms by which corticosteroids exert therapeutic benefit in COPD, reducing iron available for bacterial growth and enhancing efferocytosis.

Adrenal lesions found incidentally: how to improve clinical and cost-effectiveness.

INTRODUCTION: Adrenal incidentalomas are lesions that are incidentally identified while scanning for other conditions. While most are benign and hormonally non-functional, around 20% are malignant and/or hormonally active, requiring prompt intervention. Malignant lesions can be aggressive and life-threatening, while hormonally active tumours cause various endocrine disorders, with significant morbidity and mortality. Despite this, management of patients with adrenal incidentalomas is variable, with no robust evidence base. This project aimed to establish more effective and timely management of these patients. METHODS: We developed a web-based, electronic Adrenal Incidentaloma Management System (eAIMS), which incorporated the evidence-based and National Health Service-aligned 2016 European guidelines. The system captures key clinical, biochemical and radiological information necessary for adrenal incidentaloma patient management and generates a pre-populated outcome letter, saving clinical and administrative time while ensuring timely management plans with enhanced safety. Furthermore, we developed a prioritisation strategy, with members of the multidisciplinary team, which prioritised high-risk individuals for detailed discussion and management. Patient focus groups informed process-mapping and multidisciplinary team process re-design and patient information leaflet development. The project was partnered by University Hospital of South Manchester to maximise generalisability. RESULTS: Implementation of eAIMS, along with improvements in the prioritisation strategy, resulted in a 49% reduction in staff hands-on time, as well as a 78% reduction in the time from adrenal incidentaloma identification to multidisciplinary team decision. A health economic analysis identified a 28% reduction in costs. CONCLUSIONS: The system's in-built data validation and the automatic generation of the multidisciplinary team outcome letter improved patient safety through a reduction in transcription errors. We are currently developing the next stage of the programme to proactively identify all new adrenal incidentaloma cases.

P38 MAPK and glucocorticoid receptor crosstalk in bronchial epithelial cells.

p38 MAPK inhibition may have additive and synergistic anti-inflammatory effects when used with corticosteroids. We investigated crosstalk between p38 MAPK inhibitors and corticosteroids in bronchial epithelial cells to investigate synergistic effects on cytokine production and the molecular mechanisms involved. Effects of the p38 MAPK inhibitor BIRB-796 and dexamethasone alone and in combination on LPS, polyI:C or TNFα -induced IL-6, CXCL8 and RANTES were assessed in 16HBEs (human epithelial cell line) and on TNFα-induced IL-6 and CXCL8 in primary human epithelial cells from asthma patients and healthy controls. 16HBEs were used to assess effects of BIRB-796 alone and in combination with dexamethasone on glucocorticoid receptor (GR) activity by reporter gene assay, expression of GR target genes and nuclear localisation using Western blot. The effects of BIRB-796 on TNFα stimulated phosphorylation of p38 MAPK and GR at serine (S) 226 by Western blot. Epithelial levels of phosphorylated p38 MAPK and GR S226 were determined by immunohistochemistry in bronchial biopsies from asthma patients and healthy controls. BIRB-796 in combination with dexamethasone increased inhibition of cytokine production in a synergistic manner. Combination treatment significantly increased GR nuclear localisation compared to dexamethasone alone. BIRB-796 inhibited TNFα-induced p38 MAPK and GR S226 phosphorylation. Phosphorylated GR S226 and p38 MAPK levels were increased in bronchial epithelium of more severe asthma patients. Molecular crosstalk exists between p38 MAPK activation and GR function in human bronchial epithelial cells, which alters GR activity. Combining a p38 MAPK inhibitor and a corticosteroid may demonstrate therapeutic potential in severe asthma. KEY MESSAGES: • Combination of corticosteroid and p38 inhibitor in human bronchial epithelial cells • Combination increased cytokine inhibition synergistically and nuclear GR • p38 MAPK inhibition reduced TNFα-induced phosphorylation of GR at S226 but not S211 • Phosphorylated GRS226 and p38 is increased in bronchial epithelium in severe asthma • Combining a p38 inhibitor and a corticosteroid may be effective in asthma treatment.

The modulatory effects of the PDE4 inhibitors CHF6001 and roflumilast in alveolar macrophages and lung tissue from COPD patients.

BACKGROUND: We compared the anti-inflammatory effects of phosphodiesterase type 4 (PDE4) inhibitor roflumilast with CHF6001, a novel PDE4 inhibitor designed for inhaled administration, using human alveolar macrophages (AM) and lung tissue explants models. METHODS: AM from 13 chronic obstructive pulmonary disease (COPD) patients and 10 smoking controls and lung tissue from 7 COPD patients were stimulated with LPS following preincubation with roflumilast (0.000001-10 µM), CHF6001 (0.000001-0.1 µM), or vehicle. After 24 h, supernatants were analysed for cytokines by ELISA. The effects of both compounds on the phosphorylation and cellular localisation of cAMP response element binding protein (CREB) were assessed by immunofluorescence and Western blot analysis. Extracted RNA was used for quantitative PCR analysis of PDE4 A, B and D mRNA. RESULTS: PDE4 A, B and D expression were increased in alveolar macrophages and lung tissue of COPD patients compared to controls. Roflumilast and CHF6001 significantly reduced TNF-α production in AM and lung tissue. CHF6001 was more potent than roflumilast with lower EC50s of 0.02, 0.01 and 0.31 nM compared to 0.87, 0.47 and 10.8 nM in respective samples. PDE4 inhibition also inhibited secretion of the chemokines CCL2 and CCL4 from macrophages. Both compounds increased nuclear levels of phosphorylated CREB. CONCLUSION: PDE4 inhibitors caused a robust anti-inflammatory effect on TNF-α production from COPD AM, with inhibition of selective chemokines also observed. CHF6001 caused more potent inhibition of TNF-α production from COPD AM and lung tissue compared to roflumilast.

The effects of repeated Toll-like receptors 2 and 4 stimulation in COPD alveolar macrophages.

Background: COPD is a progressive inflammatory airway disease characterized by increased numbers of alveolar macrophages in the lungs. Bacterial colonization of the lungs is a common feature in COPD and can promote inflammation through continual and repeated Toll-like receptor (TLR) stimulation. We have studied the response of COPD alveolar macrophages to repetitive stimulation with TLR2 and TLR4 ligands. We investigated the effect of sequential stimulation with different ligands to determine whether this results in tolerance or amplification of the immune response. Methods: We stimulated alveolar macrophages from COPD patients (n=9) and smokers (n=8) with the TLR4 agonist lipopolysaccharide (LPS) or the TLR2 agonist Pam3CSK4 for 24 hours before restimulating again for 24 hours. Cytokine protein release and gene expression were investigated. Results: Repetitive stimulation of COPD and smokers macrophages with LPS for both 24-hour periods caused a reduction in tumor necrosis factor α, CCL5, and IL-10 production compared to cells that were not exposed initially to LPS. IL-6 and CXCL8 production were not significantly altered following repetitive LPS stimulation. The same pattern was observed for repeated stimulation with Pam3CSK4. Using COPD macrophages, LPS followed by Pam3CSK4 stimulation increased the levels of all cytokines compared to media followed by Pam3CSK4. Conclusion: TLR tolerance in COPD alveolar macrophages occurs after repetitive stimulation with the same TLR ligand, but this only occurs for selected cytokines. CXCL8 production is not reduced after repetitive TLR stimulation with the same ligand; this may be an important mechanism for the increased CXCL8 levels that have been observed in COPD. We showed that TLR4 stimulation followed by TLR2 stimulation does not cause tolerance, but enhances cytokine production. This may be a relevant mechanism by which bacteria cause excessive inflammation in COPD patients.

The potential clinical benefits of medicines optimisation through comprehensive geriatric assessment, carried out by secondary care geriatricians, in a general practice care setting in North Staffordshire, UK: a feasibility study.

OBJECTIVES: To evaluate the feasibility and potential clinical benefits of medicines optimisation through comprehensive geriatric assessment (CGA) of frail patients with multiple conditions, by secondary care geriatricians in a general practice care setting. METHODS: Seven general practitioner (GP) practices in one region of Stoke-on-Trent volunteered to take part. GPs selected patients (n=186) who were local permanent residents, at least 65 years old and on eight or more medications per day. Patients were sent a written invitation outlining the assessment purpose/format. Prior to patient assessments, primary care staff prepared packs detailing patient medical history, recent consultations, current medications, recent laboratory tests and social circumstances. One hour was allocated for the CGA per patient, with one of three geriatricians, to enable sufficient time to explore all relevant aspects. Assessment comprised a full history, thorough clinical examination, assessment of balance and mobility, mental function and information on home environment and support arrangements. After consultation, geriatricians made recommendations regarding further assessments, investigations or medication changes. Geriatricians entered their main findings and recommendations onto a standard template. RESULTS: In total, 687 recommendations for changes in patients' medication regimens were made for 169 (91%) patients. In 17 (9%) patients there was no recommendation to alter medications. This resulted in an average of four alterations in medication per patient. The predominant changes to medications were to stop medications (34%) or to reduce the dosage (24%). Starting a new medication represented 18% of all the medication changes. Adherence rates to geriatrician medication recommendations were 72% at 6 months and 65% at 12 months. CONCLUSIONS: CGA of older patients with complex needs, by geriatricians in a general practice care setting, is feasible. Our study demonstrated constructive collaboration between GPs and geriatricians from secondary care, suggesting further studies and clinical trials are feasible and have scope to yield beneficial outcomes.

RV568, a narrow-spectrum kinase inhibitor with p38 MAPK-α and -γ selectivity, suppresses COPD inflammation.

Novel anti-inflammatory approaches targeting chronically activated kinase pathways in chronic obstructive pulmonary disease (COPD) are needed. We evaluated RV568, a p38 mitogen-activated protein kinase-α and -γ and SRC family kinase inhibitor, in cellular and in vivo models relevant to COPD and examined its safety and efficacy in COPD patients.The anti-inflammatory activities of RV568 were tested in primary cultured monocytes, macrophages and bronchial epithelial cells and in vivo in lipopolysaccharide and cigarette smoke-exposed murine models. RV568 was evaluated in a 14-day trial in COPD patients.RV568 showed potent anti-inflammatory effects in monocytes and macrophages, which were often greater than those of corticosteroids or the p38 inhibitor Birb796. RV568 combined with corticosteroid had anti-inflammatory effects suggestive of a synergistic interaction in poly I:C-stimulated BEAS-2B cells and in the cigarette smoke model. In COPD patients, inhaled RV568 (50 µg and 100 µg) improved pre-bronchodilator forced expiratory volume in 1 s (69 mL and 48 mL respectively) and significantly reduced sputum malondialdehyde (p<0.05) compared to placebo, although there were no changes in sputum cell counts. Adverse events during RV568 and placebo treatment were similar.RV568 shows potent anti-inflammatory effects on cell and animal models relevant to COPD. RV568 was well-tolerated and demonstrated a modest clinical benefit in a 14-day COPD clinical trial.

Characterisation of lung macrophage subpopulations in COPD patients and controls.

Lung macrophage subpopulations have been identified based on size. We investigated characteristics of small and large macrophages in the alveolar spaces and lung interstitium of COPD patients and controls. Alveolar and interstitial cells were isolated from lung resection tissue from 88 patients. Macrophage subpopulation cell-surface expression of immunological markers and phagocytic ability were assessed by flow cytometry. Inflammatory related gene expression was measured. Alveolar and interstitial macrophages had subpopulations of small and large macrophages based on size and granularity. Alveolar macrophages had similar numbers of small and large cells; interstitial macrophages were mainly small. Small macrophages expressed significantly higher cell surface HLA-DR, CD14, CD38 and CD36 and lower CD206 compared to large macrophages. Large alveolar macrophages showed lower marker expression in COPD current compared to ex-smokers. Small interstitial macrophages had the highest pro-inflammatory gene expression levels, while large alveolar macrophages had the lowest. Small alveolar macrophages had the highest phagocytic ability. Small alveolar macrophage CD206 expression was lower in COPD patients compared to smokers. COPD lung macrophages include distinct subpopulations; Small interstitial and small alveolar macrophages with more pro-inflammatory and phagocytic function respectively, and large alveolar macrophages with low pro-inflammatory and phagocytic ability.

Mechanisms of corticosteroid insensitivity in COPD alveolar macrophages exposed to NTHi.

BACKGROUND: Non-typeable Haemophilus influenza (NTHi) infection is common in COPD. Corticosteroids can have limited therapeutic effects in COPD patients. NTHi causes corticosteroid insensitive cytokine production from COPD alveolar macrophages. We investigated the mechanisms by which NTHi causes corticosteroid insensitive inflammatory responses, and the effects of NTHi exposure on COPD macrophage polarisation. METHOD: Alveolar macrophages from COPD patients and controls were exposed to NTHi in conjunction with the corticosteroid dexamethasone and/or the p38 MAPK inhibitor BIRB-796. Cytokine release, GR phosphorylation and modulation and macrophage phenotype were analysed. RESULTS: Dexamethasone significantly inhibited NTHi induced TNF-α, IL-6 and IL-10 from COPD macrophages but, CXCL8 was not suppressed. BIRB-796 combined with dexamethasone caused significantly greater inhibition of all cytokines than either drug alone (p < 0.05 all comparisons). NTHi caused phosphorylation of GR S226 reducing GR nuclear localisation, an effect regulated by p38 MAPK. NTHi altered macrophage polarisation by increasing IL-10 and decreasing CD36, CD206, CD163 and HLA-DR. CONCLUSION: NTHi exposure causes p38 MAPK dependent GR phosphorylation associated with decreased GR function in COPD alveolar macrophages. Combining a p38 MAPK inhibitor with corticosteroids can enhance anti-inflammatory effects during NTHi exposure of COPD alveolar macrophages. NTHi causes macrophage polarisation that favours bacterial persistence.

Characterization of TLR-induced inflammatory responses in COPD and control lung tissue explants.

PURPOSE: Viruses are a common cause of exacerbations in chronic obstructive pulmonary disease (COPD). They activate toll-like receptors (TLRs) 3, 7, and 8, leading to a pro-inflammatory response. We have characterized the responses of TLR3 and TLR7/8 in lung tissue explants from COPD patients and control smokers. METHODS: We prepared lung whole tissue explants (WTEs) from patients undergoing surgery for confirmed or suspected lung cancer. In order to mimic the conditions of viral infection, we used poly(I:C) for TLR3 stimulation and R848 for TLR7/8 stimulation. These TLR ligands were used alone and in combination. The effects of tumor necrosis factor α (TNFα) neutralization and dexamethasone on TLR responses were examined. Inflammatory cytokine release was measured by enzyme-linked immunosorbent assay and gene expression by quantitative real-time polymerase chain reaction. RESULTS: WTEs from COPD patients released higher levels of pro-inflammatory cytokines compared with WTEs from smokers. Activation of multiple TLRs led to a greater than additive release of TNFα and CCL5. TNFα neutralization and dexamethasone treatment decreased cytokine release. CONCLUSION: This WTE model shows an enhanced response of COPD compared with controls, suggesting an increased response to viral infection. There was amplification of innate immune responses with multiple TLR stimulation.

Neutral sphingomyelinase-2, acid sphingomyelinase, and ceramide levels in COPD patients compared to controls.

BACKGROUND: Increased pulmonary ceramide levels are suggested to play a causative role in lung diseases including COPD. Neutral sphingomyelinase-2 (nSMase-2) and acid SMase (aSMase), which hydrolyze sphingomyelin to produce ceramide, are activated by a range of cellular stresses, including inflammatory cytokines and pathogens, but notably cigarette smoke appears to only activate nSMase-2. Our primary objective was to investigate nSMase-2 and aSMase protein localization and quantification in lung tissue from nonsmokers (NS), smokers (S), and COPD patients. In addition, various ceramide species (C16, C18, and C20) were measured in alveolar macrophages from COPD patients versus controls. MATERIALS AND METHODS: Patients undergoing surgical resection for suspected or confirmed lung cancer were recruited, and nSMase-2 and aSMase protein was investigated in different areas of lung tissue (small airways, alveolar walls, subepithelium, and alveolar macrophages) by immunohistochemistry. Ceramide species were measured in alveolar macrophages from COPD patients and controls by mass spectrometry. RESULTS: nSMase-2 and aSMase were detected in the majority of small airways. There was a significant increase in nSMase-2 immunoreactivity in alveolar macrophages from COPD patients (54%) compared with NS (31.7%) (P<0.05), and in aSMase immunoreactivity in COPD (68.2%) and S (69.5%) alveolar macrophages compared with NS (52.4%) (P<0.05). aSMase labeling was also increased in the subepithelium and alveolar walls of S compared with NS. Ceramide (C20) was significantly increased in alveolar macrophages from COPD patients compared with controls. CONCLUSION: nSMase-2 and aSMase are both increased in COPD alveolar macrophages at the protein level; this may contribute toward the elevated ceramide (C20) detected in alveolar macrophages from COPD patients.