Activation of the Complement and Coagulation Systems in the Small Airways in Asthma.

BACKGROUND: Several studies have shown the importance of the complement and coagulation systems in the pathogenesis of asthma. OBJECTIVES: We explored whether we could detect differentially abundant complement and coagulation proteins in the samples obtained from the small airway lining fluid by collection of exhaled particles in patients with asthma and whether these proteins are associated with small airway dysfunction and asthma control. METHOD: Exhaled particles were obtained from 20 subjects with asthma and 10 healthy controls (HC) with the PExA method and analysed with the SOMAscan proteomics platform. Lung function was assessed by nitrogen multiple breath washout test and spirometry. RESULTS: 53 proteins associated with the complement and coagulation systems were included in the analysis. Nine of those proteins were differentially abundant in subjects with asthma as compared to HC, and C3 was significantly higher in inadequately controlled asthma as compared to well-controlled asthma. Several proteins were associated with physiological tests assessing small airways. CONCLUSIONS: The study highlights the role of the local activation of the complement and coagulation systems in the small airway lining fluid in asthma and their association with both asthma control and small airway dysfunction. The findings highlight the potential of complement factors as biomarkers to identify different sub-groups among patients with asthma that could potentially benefit from a therapeutic approach targeting the complement system.

Differential Contribution of NF-κB Signaling Pathways to CD4+ Memory T Cell Induced Activation of Endothelial Cells.

Endothelial cells (ECs) are important contributors to inflammation in immune-mediated inflammatory diseases (IMIDs). In this study, we examined whether CD4+ memory T (Tm) cells can drive EC inflammatory responses. Human Tm cells produced ligands that induced inflammatory responses in human umbilical vein EC as exemplified by increased expression of inflammatory mediators including chemokines and adhesion molecules. NF-κB, a key regulator of EC activation, was induced by Tm cell ligands. We dissected the relative contribution of canonical and non-canonical NF-κB signaling to Tm induced EC responses using pharmacological small molecule inhibitors of IKKß (iIKKß) or NF-κB inducing kinase (iNIK). RNA sequencing revealed substantial overlap in IKKß and NIK regulated genes (n=549) that were involved in inflammatory and immune responses, including cytokines (IL-1ß, IL-6, GM-CSF) and chemokines (CXCL5, CXCL1). NIK regulated genes were more restricted, as 332 genes were uniquely affected by iNIK versus 749 genes by iIKKß, the latter including genes involved in metabolism, proliferation and leukocyte adhesion (VCAM-1, ICAM-1). The functional importance of NIK and IKKß in EC activation was confirmed by transendothelial migration assays with neutrophils, demonstrating stronger inhibitory effects of iIKKß compared to iNIK. Importantly, iIKKß - and to some extent iNIK - potentiated the effects of currently employed therapies for IMIDs, like JAK inhibitors and anti-IL-17 antibodies, on EC inflammatory responses. These data demonstrate that inhibition of NF-κB signaling results in modulation of Tm cell-induced EC responses and highlight the potential of small molecule NF-κB inhibitors as a novel treatment strategy to target EC inflammatory responses in IMIDs.

A novel non-invasive method allowing for discovery of pathologically relevant proteins from small airways.

BACKGROUND: There is a lack of early and precise biomarkers for personalized respiratory medicine. Breath contains an aerosol of droplet particles, which are formed from the epithelial lining fluid when the small airways close and re-open during inhalation succeeding a full expiration. These particles can be collected by impaction using the PExA® method (Particles in Exhaled Air), and are derived from an area of high clinical interest previously difficult to access, making them a potential source of biomarkers reflecting pathological processes in the small airways. RESEARCH QUESTION: Our aim was to investigate if PExA method is useful for discovery of biomarkers that reflect pathology of small airways. METHODS AND ANALYSIS: Ten healthy controls and 20 subjects with asthma, of whom 10 with small airway involvement as indicated by a high lung clearance index (LCI ≥ 2.9 z-score), were examined in a cross-sectional design, using the PExA instrument. The samples were analysed with the SOMAscan proteomics platform (SomaLogic Inc.). RESULTS: Two hundred-seven proteins were detected in up to 80% of the samples. Nine proteins showed differential abundance in subjects with asthma and high LCI as compared to healthy controls. Two of these were less abundant (ALDOA4, C4), and seven more abundant (FIGF, SERPINA1, CD93, CCL18, F10, IgM, IL1RAP). sRAGE levels were lower in ex-smokers (n = 14) than in never smokers (n = 16). Gene Ontology (GO) annotation database analyses revealed that the PEx proteome is enriched in extracellular proteins associated with extracellular exosome-vesicles and innate immunity. CONCLUSION: The applied analytical method was reproducible and allowed identification of pathologically interesting proteins in PEx samples from asthmatic subjects with high LCI. The results suggest that PEx based proteomics is a novel and promising approach to study respiratory diseases with small airway involvement.

Plasma proteins elevated in severe asthma despite oral steroid use and unrelated to Type-2 inflammation.

RATIONALE: Asthma phenotyping requires novel biomarker discovery. OBJECTIVES: To identify plasma biomarkers associated with asthma phenotypes by application of a new proteomic panel to samples from two well-characterised cohorts of severe (SA) and mild-to-moderate (MMA) asthmatics, COPD subjects and healthy controls (HCs). METHODS: An antibody-based array targeting 177 proteins predominantly involved in pathways relevant to inflammation, lipid metabolism, signal transduction and extracellular matrix was applied to plasma from 525 asthmatics and HCs in the U-BIOPRED cohort, and 142 subjects with asthma and COPD from the validation cohort BIOAIR. Effects of oral corticosteroids (OCS) were determined by a 2-week, placebo-controlled OCS trial in BIOAIR, and confirmed by relation to objective OCS measures in U-BIOPRED. RESULTS: In U-BIOPRED, 110 proteins were significantly different, mostly elevated, in SA compared to MMA and HCs. 10 proteins were elevated in SA versus MMA in both U-BIOPRED and BIOAIR (alpha-1-antichymotrypsin, apolipoprotein-E, complement component 9, complement factor I, macrophage inflammatory protein-3, interleukin-6, sphingomyelin phosphodiesterase 3, TNF receptor superfamily member 11a, transforming growth factor-ß and glutathione S-transferase). OCS treatment decreased most proteins, yet differences between SA and MMA remained following correction for OCS use. Consensus clustering of U-BIOPRED protein data yielded six clusters associated with asthma control, quality of life, blood neutrophils, high-sensitivity C-reactive protein and body mass index, but not Type-2 inflammatory biomarkers. The mast cell specific enzyme carboxypeptidase A3 was one major contributor to cluster differentiation. CONCLUSIONS: The plasma proteomic panel revealed previously unexplored yet potentially useful Type-2-independent biomarkers and validated several proteins with established involvement in the pathophysiology of SA.

Smoking induces sex-specific changes in the small airway proteome.

INTRODUCTION: Cigarette smoke triggers many cellular and signaling responses in the lung and the resulting inflammation plays a central role in smoke-related lung diseases, such as COPD. We explored the effects of smoking on the small airway proteome in samples obtained by collection of exhaled particles with the aim to identify specific proteins dysregulated by smoking. METHODS: Exhaled particles were obtained from 38 current smokers, 47 former smokers and 22 healthy controls with the PExA method. 120 ng of sample was collected from individual subjects and analyzed with the SOMAscan proteomics platform. General linear model-based statistics were performed. RESULTS: Two hundred and three proteins were detected in at least half of 107 total samples. Active smoking exerted a significant impact on the protein composition of respiratory tract lining fluid (RTLF), with 81 proteins altered in current smokers compared to never smokers (p < 0.05, q < 0.124). Among the proteins most clearly discriminating between current and never smokers were sRAGE, FSTL3, SPOCK2 and protein S, all of them being less abundant in current smokers. Analysis stratified for sex unveiled sex differences with more pronounced proteomic alterations due to active smoking in females than males. Proteins whose abundance was altered by active smoking in women were to a larger extent related to the complement system. The small airway protein profile of former smokers appeared to be more similar to that observed in never smokers. CONCLUSIONS: The study shows that smoking has a strong impact on protein expression in the small airways, and that smoking affects men and women differently, suggesting PExA sampling combined with high sensitivity protein analysis offers a promising platform for early detection of COPD and identification of novel COPD drug targets.

Impaired Butyrate Induced Regulation of T Cell Surface Expression of CTLA-4 in Patients with Ulcerative Colitis.

Patients with ulcerative colitis (UC) have reduced intestinal levels of short-chain fatty acids (SCFAs), including butyrate, which are important regulators of host-microbiota crosstalk. The aim was therefore to determine effects of butyrate on blood and intestinal T cells from patients with active UC. T cells from UC patients and healthy subjects were polyclonally stimulated together with SCFAs and proliferation, activation, cytokine secretion, and surface expression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) were analyzed. Butyrate induced comparable, dose dependent inhibition of activation and proliferation in blood T cells and activation in intestinal T cells from UC patients and healthy subjects. However, surface expression of the inhibitory molecule CTLA-4 on stimulated blood and intestinal T cells was impaired in UC patients and was not restored following butyrate treatment. Furthermore, unlike intestinal T cells from healthy subjects, butyrate was unable to downregulate secretion of interferon gamma (IFNγ), interleukin (IL)-4, IL-17A, and IL-10 in UC patients. Although seemingly normal inhibitory effects on T cell activation and proliferation, butyrate has an impaired ability to reduce cytokine secretion and induce surface expression of CTLA-4 in T cells from UC patients with active disease. Overall, these observations indicate a dysfunction in butyrate induced immune regulation linked to CTLA-4 signaling in T cells from UC patients during a flare.

Within-session reproducibility of forced oscillometry.

BACKGROUND: The forced oscillation technique (FOT) provides detailed information about the mechanics of the respiratory system, while requiring minimal co-operation by the patient. FOT may be abnormal in subjects with normal spirometry and appears to be more closely related to airway symptoms. It is, therefore, attractive in epidemiological studies, where a large number of different examinations are made in each subjects in a short period of time. Current technical standards recommend the mean of three consecutive measurements to be used, but there is limited information regarding within-session variability of FOT measurements. OBJECTIVE: The purpose of this study was to examine the within-session variability in FOT measurements in a large, population-based sample. METHODS: We performed three consecutive FOT measurements in 700 subjects using the impulse oscillometry system. The first measurement was compared to the mean of three measurements for resistance at 5 and 20 Hz (R5 and R20, respectively), R5-R20, reactance at 5 Hz (X5) and resonant frequency (fres ). RESULTS: The differences between the first and the mean of three measurements (median, interquartile range) were minimal, for example 0.002, -0.008 to 0.014 kPa L-1  s for R5 and -0.001, -0.008 to 0.005 kPa L-1  s for X5. Findings were numerically similar for men and women as well as for subjects with and without airflow obstruction at spirometry. CONCLUSIONS: We conclude that, whereas in clinical situations, three FOT measurements are to be preferred, a single measurement may suffice in epidemiological studies.

Multi-omics links IL-6 trans-signalling with neutrophil extracellular trap formation and Haemophilus infection in COPD.

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-1ß, IL-8, macrophage inflammatory protein-1ß) 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 by Haemophilus influenzae were 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, with Haemophilus as the dominating genus. NETosis induced by H. influenzae was 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 with Haemophilus may be an important disease driver in a subset of COPD patients.

Back to the future: re-establishing guinea pig in vivo asthma models.

Research using animal models of asthma is currently dominated by mouse models. This has been driven by the comprehensive knowledge on inflammatory and immune reactions in mice, as well as tools to produce genetically modified mice. Many of the identified therapeutic targets influencing airway hyper-responsiveness and inflammation in mouse models, have however been disappointing when tested clinically in asthma. It is therefore a great need for new animal models that more closely resemble human asthma. The guinea pig has for decades been used in asthma research and a comprehensive table of different protocols for asthma models is presented. The studies have primarily been focused on the pharmacological aspects of the disease, where the guinea pig undoubtedly is superior to mice. Further reasons are the anatomical and physiological similarities between human and guinea pig airways compared with that of the mouse, especially with respect to airway branching, neurophysiology, pulmonary circulation and smooth muscle distribution, as well as mast cell localization and mediator secretion. Lack of reagents and specific molecular tools to study inflammatory and immunological reactions in the guinea pig has however greatly diminished its use in asthma research. The aim in this position paper is to review and summarize what we know about different aspects of the use of guinea pig in vivo models for asthma research. The associated aim is to highlight the unmet needs that have to be addressed in the future.

NIK-IKK complex interaction controls NF-κB-dependent inflammatory activation of endothelium in response to LTßR ligation.

NF-κB-inducing kinase (NIK; also known as MAP3K14) is a central regulator of non-canonical NF-κB signaling in response to stimulation of TNF receptor superfamily members, such as the lymphotoxin-ß receptor (LTßR), and is implicated in pathological angiogenesis associated with chronic inflammation and cancer. Here, we identify a previously unrecognized role of the LTßR-NIK axis during inflammatory activation of human endothelial cells (ECs). Engagement of LTßR-triggered canonical and non-canonical NF-κB signaling promoted expression of inflammatory mediators and adhesion molecules, and increased immune cell adhesion to ECs. Sustained LTßR-induced inflammatory activation of ECs was NIK dependent, but independent of p100, indicating that the non-canonical arm of NF-κB is not involved. Instead, prolonged activation of canonical NF-κB signaling, through the interaction of NIK with IκB kinase α and ß (also known as CHUK and IKBKB, respectively), was required for the inflammatory response. Endothelial inflammatory activation induced by synovial fluid from rheumatoid arthritis patients was significantly reduced by NIK knockdown, suggesting that NIK-mediated alternative activation of canonical NF-κB signaling is a key driver of pathological inflammatory activation of ECs. Targeting NIK could thus provide a novel approach for treating chronic inflammatory diseases.