Background:Interleukin (IL)-6 trans-signalling (IL-6TS) is emerging as a pathogenic mechanism in chronic respiratory diseases; however, the drivers of IL-6TS in the airways and the phenotypic characteristic of patients with increased IL-6TS pathway activation remain poorly understood.Objective:Our aim was to identify and characterise COPD patients with increased airway IL-6TS and to elucidate the biological drivers of IL-6TS pathway activation.Methods:We used an IL-6TS-specific sputum biomarker profile (soluble IL-6 receptor (sIL-6R), IL-6, IL-1β, IL-8, macrophage inflammatory protein-1β) to stratify sputum data from patients with COPD (n=74; Biomarkers to Target Antibiotic and Systemic Corticosteroid Therapy in COPD Exacerbation (BEAT-COPD)) by hierarchical clustering. The IL-6TS signature was related to clinical characteristics and sputum microbiome profiles. The induction of neutrophil extracellular trap formation (NETosis) and IL-6TS byHaemophilus influenzaewere studied in human neutrophils.Results:Hierarchical clustering revealed an IL-6TS-high subset (n=24) of COPD patients, who shared phenotypic traits with an IL-6TS-high subset previously identified in asthma. The subset was characterised by increased sputum cell counts (p=0.0001), persistent sputum neutrophilia (p=0.0004), reduced quality of life (Chronic Respiratory Questionnaire total score; p=0.008), and increased levels of pro-inflammatory mediators and matrix metalloproteinases in sputum. IL-6TS-high COPD patients showed an increase in Proteobacteria, withHaemophilusas the dominating genus. NETosis induced byH. influenzaewas identified as a potential mechanism for increased sIL-6R levels. This was supported by a significant positive correlation between sIL-6R and NETosis markers in bronchoalveolar lavage fluid from COPD patients.Conclusion:IL-6TS pathway activation due to chronic colonisation withHaemophilusmay be an important disease driver in a subset of COPD patients.

Introduction and aim: Emerging evidence suggests that NLR pyrin domain-containing protein 3 (NLRP3) inflammasome activation may be persistent in the airways and contribute to inflammation and pathological cell damage in COPD. The aim of this study was to identify and characterize COPD patients with increased inflammasome activation. Methods: Inflammasome-induced pro-inflammatory cytokines IL-1β and IL-18 were measured in bronchoalveolar lavage fluid (BALF) from COPD patients (n=29), healthy non-smokers (n=27) and smokers (n=8). The inflammasome-high cluster of COPD patients, identified by hierarchical clustering of IL-1β (total and active form) and IL-18, was related to clinical characteristics and disease biomarkers in BALF. Results: The levels of inflammasome cytokines were significantly higher in BALF from COPD patients compared to healthy volunteers. The inflammasome-high cluster of COPD patients (48%) was distinguished by increased sputum neutrophils (p=0.03), decreased eosinophils (p=0.01) and increased bacterial colonization (p =0.04). Additionally, the inflammasome-high cluster exhibited significantly increased BALF levels of surrogate neutrophil extracellular traps (NETosis) markers; cell free DNA (p<0.001) and Myeloperoxidase (MPO; p <0.001), compared to inflammasome-low patients. Conclusions: Inflammasome activation is increased in the airways of COPD patients with increased neutrophilic inflammation and bacterial colonization. Recent findings revealed an interplay between NLRP3 inflammasome activation and NETosis in several disorders. Our results are in line with these findings, warranting further investigation of the interactions between NLRP3 inflammasome and NETosis in COPD.

Background: p38 mitogen-activated protein kinase (MAPK) plays a central role in the regulation and activation of pro-inflammatory mediators.COPD patients have increased levels of activated p38 MAPK, which correlate with increased lung function impairment, alveolar wall inflammation, and COPD exacerbations.Objectives: These studies aimed to assess the effect of p38 inhibition with AZD7624 in healthy volunteers and patients with COPD.The principal hypothesis was that decreasing lung inflammation via inhibition of p38α would reduce exacerbations and improve quality of life for COPD patients at high risk for acute exacerbations.Methods: The p38 isoform most relevant to lung inflammation was assessed using an in situ proximity ligation assay in severe COPD patients and donor controls.Volunteers aged 18-55 years were randomized into the lipopolysaccharide (LPS) challenge study, which investigated the effect of a single dose of AZD7624 vs placebo on inflammatory biomarkers.The Proof of Principle study randomized patients aged 40-85 years with a diagnosis of COPD for .1 year to AZD7624 or placebo to assess the effect of p38 inhibition in decreasing the rate of exacerbations.Results: The p38 isoform most relevant to lung inflammation was p38α, and AZD7624 specifically inhibited p38α and p38β isoforms in human alveolar macrophages.Thirty volunteers were randomized in the LPS challenge study.AZD7624 reduced the increase from baseline in sputum neutrophils and TNF-α by 56.6% and 85.4%, respectively (p,0.001).In the 213 patients randomized into the Proof of Principle study, there was no statistically significant difference between AZD7624 and placebo when comparing the number of days to the first moderate or severe exacerbation or early dropout.Conclusion: Although p38α is upregulated in the lungs of COPD patients, AZD7624, an isoform-specific inhaled p38 MAPK inhibitor, failed to show any benefit in patients with COPD.

Inhaled drugs generally aim to drive a local pharmacological effect in lung, at the same time minimizing systemic exposure, in order to obtain efficacy in lung disease without unwanted systemic effects. Here, we demonstrate that inhaled delivery of a p38 inhibitor (AZD7624) can provide superior pharmacokinetic exposure and superior pharmacodynamic lung effects. In rats, inhaled AZD7624 had a five times higher dose-adjusted lung exposure compared with intravenous dosing. In healthy volunteers, lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) in sputum has been shown to be significantly reduced (85%) by means of inhaled AZD7624. Here, we demonstrate that this effect is associated with a mean unbound plasma concentration, gained from in vitro and ex vivo LPS-challenge protocols, significantly below potencies obtained for AZD7624, suggesting that lung exposure is probably much higher than systemic exposure. This assessment was made for the unbound potency (pIC50u), e.g., the potency remaining after adjustment for plasma protein binding and blood plasma ratio. Hence, the unbound potency of AZD7624 to inhibit LPS-induced TNFα in human mononuclear cells, in whole blood as well as in alveolar macrophages in vitro, was 8.4, 8.7 (full inhibition), and 9.0 (partial inhibition), respectively. The pIC50u in whole blood ex vivo was 8.8, showing good in vitro/in vivo potency correlations. Thus, a mean unbound AZD7624 plasma concentration of 0.3 nmol/l, which was associated with a decrease in LPS-induced sputum TNFα by 85%, is much lower than the pIC50u. This demonstrates that AZD7624 can achieve significant local lung pharmacodynamic effects with concomitant sub-pharmacological systemic exposure.

AZD7624 is an inhaled p38 inhibitor being developed for COPD. Here we used LPS challenge in i) in vitro blood derived mononuclear cells (MC) and alveolar macrophages (AM), ii) ex vivo human blood and iii) in vivo inhaled LPS in order to determine both the translatability of unbound potency (nM) between methods and if the effect of AZD7624 in the lung (potency) versus systemic potency is enhanced by inhalation. In vitro, cells were pretreated with AZD7624 prior to LPS. Ex-vivo, human blood was obtained from subjects having inhaled AZD7624, prior to LPS. In vivo, rats were given inhaled AZD7624 at different doses at different time-points prior to an inhaled LPS challenge. TNFa was determined in lung lavage. Healthy volunteers were given inhaled AZD7624 prior to inhaled LPS, followed by sputum induction in which TNFa was measured. In vitro, the potency was 4 nM (human MC and whole blood) and 3-7/0.5 nM (rat/human, AM). Ex-vivo, the potency was 2 nM when AZD7624 was given by inhalation. In vivo in rats, inhaled AZD7624 decreased lung TNFa response to inhaled LPS with mean plasma concentrations In different models, there is a consistency with similar potencies of AZD7624. The in vivo potency of inhaled AZD7624 on LPS induced TNFa in lungs tends to be higher than on LPS induced TNFa in vitro and ex-vivo in blood. This implies that inhalation of AZD7624 (and thus higher local exposure) provides a benefit by an increased local anti-inflammatory effect compared to oral delivery systemic activity.

Inhaled LPS Challenge has been used to test compounds for COPD. Systemic markers of inflammation, (e.g. neutrophil count, IL-6 levels, and CRP), are associated with risk of acute exacerbation of COPD. Whether an inhaled anti-inflammatory might affect these systemic markers, and the relationship between these markers in lung and serum is unclear. Here we present data on the effect AZD7624, an inhaled p38 inhibitor for COPD, on lung and systemic markers of inflammation after LPS challenge. 30 subjects were randomized in a double-blind, placebo-controlled cross-over study Subjects received a single inhaled dose of AZD7624 (1200 mcg) or placebo 30 minutes prior to LPS challenge, followed by sputum induction 6 hours post challenge (6.5h post dose) and blood sampling at 0.25, 6.5, 12 and 24h post dose for measurement of cell counts and inflammatory biomarkers. AZD7624 attenuated sputum neutrophil differential increase after LPS challenge by 56.8% (P In conclusion, inhaled AZD7624 significantly inhibits LPS-induced lung and systemic inflammation, and lung inflammation markers correlate with systemic markers indicating a potential for AZD7624 as an inhaled treatment for COPD.

GPR120 (Ffar4) has been postulated to represent an important receptor mediating the improved metabolic profile seen upon ingestion of a diet enriched in polyunsaturated fatty acids (PUFAs). GPR120 is highly expressed in the digestive system, adipose tissue, lung and macrophages and also present in the endocrine pancreas. A new Gpr120 deficient mouse model on pure C57bl/6N background was developed to investigate the importance of the receptor for long-term feeding with a diet enriched with fish oil. Male Gpr120 deficient mice were fed two different high fat diets (HFDs) for 18 weeks. The diets contained lipids that were mainly saturated (SAT) or mainly n-3 polyunsaturated fatty acids (PUFA). Body composition, as well as glucose, lipid and energy metabolism, was studied. As expected, wild type mice fed the PUFA HFD gained less body weight and had lower body fat mass, hepatic lipid levels, plasma cholesterol and insulin levels and better glucose tolerance as compared to those fed the SAT HFD. Gpr120 deficient mice showed a similar improvement on the PUFA HFD as was observed for wild type mice. If anything, the Gpr120 deficient mice responded better to the PUFA HFD as compared to wild type mice with respect to liver fat content, plasma glucose levels and islet morphology. Gpr120 deficient animals were found to have similar energy, glucose and lipid metabolism when fed HFD PUFA compared to wild type mice. Therefore, GPR120 appears to be dispensable for the improved metabolic profile associated with intake of a diet enriched in n-3 PUFA fatty acids.

Elevated circulating levels of secretory phospholipase A(2) (sPLA(2)) are associated with atherosclerotic cardiovascular disease. sPLA(2) can contribute to atherogenesis by hydrolyzing phospholipids of circulating lipoproteins and lipoproteins entrapped in the arterial wall and/or in cells that reside in the intima and that participate in the inflammatory response to lipoprotein deposition. This article reviews differences and similarities between sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X, all of which are members of this family of enzymes with reported potential proatherogenic features. Published data suggest that each of the enzymes has a distinct profile characterized by differences in tissue expression and localization, capacity to act on phospholipids of cell membranes and lipoproteins, and their interaction with arterial proteoglycans. In addition, the article discusses results from the authors' laboratory showing that diet-induced or gene-induced hyperlipidemia in mice enhances the expression of sPLA(2)-V in different tissues, but not sPLA(2)-IIA. Such differences indicate that these enzymes may have different roles in atherosclerotic cardiovascular disease through their distinct profiles.