Dr Michael Fricker

Background and objective: Chronic obstructive pulmonary disease (COPD) is the third leading cause of illness and death worldwide. Current treatments aim to control symptoms with none able to reverse disease or stop its progression. We explored the major molecular changes in COPD pathogenesis. Methods: We employed quantitative label-based proteomics to map the changes in the lung tissue proteome of cigarette smoke-induced experimental COPD that is induced over 8 weeks and progresses over 12 weeks. Results: Quantification of 7324 proteins enabled the tracking of changes to the proteome. Alterations in protein expression profiles occurred in the induction phase, with 18 and 16 protein changes at 4- and 6-week time points, compared to age-matched controls, respectively. Strikingly, 269 proteins had altered expression after 8 weeks when the hallmark pathological features of human COPD emerge, but this dropped to 27 changes at 12 weeks with disease progression. Differentially expressed proteins were validated using other mouse and human COPD bronchial biopsy samples. Major changes in RNA biosynthesis (heterogeneous nuclear ribonucleoproteins C1/C2 [HNRNPC] and RNA-binding protein Musashi homologue 2 [MSI2]) and modulators of inflammatory responses (S100A1) were notable. Mitochondrial dysfunction and changes in oxidative stress proteins also occurred. Conclusion: We provide a detailed proteomic profile, identifying proteins associated with the pathogenesis and disease progression of COPD establishing a platform to develop effective new treatment strategies.

Purpose: Systemic inflammatory biomarkers can improve diagnosis and assessment of chronic obstructive pulmonary disease (COPD) and asthma. We aimed to validate an airway disease biomarker panel of 4 systemic inflammatory biomarkers, a2-macroglobulin, ceruloplasmin, haptoglobin and hemopexin, to establish their relationship to airway disease diagnosis and inflammatory phenotypes and to identify an optimized biomarker panel for disease differentiation. Methods: Participants with COPD or asthma were classified by inflammatory phenotypes. Immunoassay methods were used to measure levels of validation biomarkers in the sera of participants with disease and non-respiratory disease controls. Markers were analyzed individually and in combination for disease differentiation and compared to established biomarkers (C-reactive protein, interleukin-6, and white blood cell/blood eosinophil count). Results: The study population comprised of 141 COPD, 127 severe asthma, 54 mild-moderate asthma and 71 control participants. Significant differences in ceruloplasmin, haptoglobin and hemopexin levels between disease groups and between systemic inflammatory phenotypes were observed. However, no differences were found between airway inflammatory phenotypes. Hemopexin was the best performing individual biomarker and could diagnose COPD versus control participants (area under the curve [AUC], 98.3%; 95% confidence interval [CI], 96.7%-99.9%) and differentiate COPD from asthmatic participants (AUC, 97.0%; 95% CI, 95.4%-98.6%), outperforming established biomarkers. A biomarker panel, including hemopexin, haptoglobin and other established biomarkers, could diagnose asthma versus control participants (AUC, 87.5%; 95% CI, 82.8%-92.2%). Conclusions: Hemopexin can be a novel biomarker with superior diagnostic ability in differentiating COPD and asthma. We propose an anti-inflammatory axis between the airways and systemic circulation, in which hemopexin is a protective component in airway disease.

Background and objective: Severe asthma is responsible for a disproportionate burden of illness and healthcare costs spent on asthma. This study analyses sputum transcriptomics to investigate the mechanisms and novel treatment targets of severe asthma. Methods: Induced sputum samples were collected in a cross-sectional study from participants with severe asthma (n = 12, defined as per GINA criteria), non-severe uncontrolled (n = 21) and controlled asthma (n = 21) and healthy controls (n = 15). Sputum RNA was extracted and transcriptomic profiles were generated (Illumina HumanRef-8 V2) and analysed (GeneSpring). Sputum protein lysates were analysed for p38 activation in a validation study (n = 24 asthma, n = 8 healthy) by western blotting. Results: There were 2166 genes differentially expressed between the four groups. In severe asthma, the expression of 1875, 1308 and 563 genes was altered compared to healthy controls, controlled and uncontrolled asthma, respectively. Of the 1875 genes significantly different to healthy controls, 123 were >2-fold change from which four networks were identified. Thirty genes (>2-fold change) were significantly different in severe asthma compared to both controlled asthma and healthy controls. There was enrichment of genes in the p38 signalling pathway that were associated with severe asthma. Phosphorylation of p38 was increased in a subset of severe asthma samples, correlating with neutrophilic airway inflammation. Conclusion: Severe asthma is associated with substantial differences in sputum gene expression that underlie unique cellular mechanisms. The p38 signalling pathway may be important in the pathogenesis of severe asthma, and future investigations into p38 inhibition are warranted as a ¿non-Th2¿ therapeutic option.

Background: The 6 gene expression signature (6GS) predicts in¿ammatory phenotype, exacerbation risk, and corticosteroid responsiveness in asthma. In COPD, patterns of airway in¿ammation are similar, suggesting the 6GS may be useful. This study determines the diagnostic and prognostic ability of 6GS in predicting in¿ammatory phenotypes and exacerbation risk in COPD. Methods: We performed 2 studies: a cross-sectional phenotype prediction study in stable COPD (total N=132; n=34 eosinophilic (E)-COPD, n=42 neutrophilic (N)-COPD, n=39 paucigranulocytic (PG)-COPD, n=17 mixed-granulocytic (MG)-COPD) that assessed 6GS ability to discriminate phenotypes (eosinophilia=3%; neutrophilia=61%); and a prospective cohort study (total n=54, n=8 E-COPD; n=18 N-COPD; n=20 PG-COPD; n=8 MG-COPD, n=21 exacerbation prone (=2/year)) that investigated phenotype and exacerbation prediction utility. 6GS was measured by qPCR and evaluated using multiple logistic regression and area under the curve (AUC). Short-term reproducibility (intra-class correlation) and phenotyping method agreement (¿ statistic) were assessed. Results: In the phenotype prediction study, 6GS could accurately identify and discriminate patients with E-COPD from N-COPD (AUC=96.4%; p<0.0001), PG-COPD (AUC=88.2%; p<0.0001) or MG-COPD (AUC=86.2%; p=0.0001), as well as N-COPD from PG-COPD (AUC=83.6%; p<0.0001) or MG-COPD (AUC=87.4%; p<0.0001) and was reproducible. In the prospective cohort study, 6GS had substantial agreement for neutrophilic in¿ammation (82%, ¿=0.63,p<0.001)and moderate agreement foreosinophilici n¿ammation(78%, ¿=0.42,p<0.001). 6GS could signi¿cantly discriminate exacerbationprone patients (AUC=77.2%; p=0.034). Higher IL1B levels were associated with poorer lung function and increased COPD severity. Conclusion: 6GS can signi¿cantly and reproducibly discriminate COPD in¿ammatory phenotypes and predict exacerbation prone patients and may become a useful molecular diagnostic tool assisting COPD management..

Background: Improved diagnostic tools for predicting future exacerbation frequency in asthmatic patients are required. A sputum gene expression signature of 6 biomarkers (6-gene signature [6GS], including Charcot-Leyden crystal galectin [CLC]; carboxypeptidase 3 [CPA3]; deoxyribonuclease 1-like 3 [DNASE1L3]; alkaline phosphatase, liver/bone/kidney [ALPL]; CXCR2; and IL1B) predicts inflammatory and treatment response phenotypes in patients with stable asthma. Recently, we demonstrated that azithromycin (AZM) add-on treatment in patients with uncontrolled moderate-to-severe asthma significantly reduced asthma exacerbations (AMAZES clinical trial). Objectives: We sought to test whether the 6GS predicts future exacerbation and inflammatory phenotypes in a subpopulation of AMAZES and to test the effect of AZM therapy on 6GS expression and prognostic capacity. Methods: One hundred forty-two patients (73 placebo-treated and 69 AZM-treated patients) had sputum stored for quantitative PCR of 6GS markers at baseline and after 48 weeks of treatment. Logistic regression and receiver operating characteristic and area under the curve (AUC) determination were performed on baseline measures, and in an exploratory analysis the predictive value of the 6GS was compared with conventional biomarkers for exacerbation and inflammatory phenotypes. Results: The 6GS significantly predicted all future exacerbation phenotypes tested. Calculated AUCs for the 6GS were significantly greater than AUCs for peripheral blood eosinophil counts, sputum neutrophil counts, and combined sputum eosinophil and neutrophil counts. 6GS AUCs were also numerically but not significantly greater than those for fractional exhaled nitric oxide values and sputum eosinophil counts. AZM treatment altered neither 6GS expression nor the predictive capacity of the 6GS for future exacerbation phenotypes. The 6GS was a significant predictor of airway inflammatory phenotype in this population. Conclusion: We demonstrate that a sputum gene signature can predict future exacerbation phenotypes of asthma, with the greatest biomarker performance in identifying those who would experience frequent severe exacerbations. AZM therapy did not modify 6GS expression or biomarker performance, suggesting the therapeutic action of AZM is independent of 6GS-related inflammatory pathways.

Background: Dysfunction of the bronchial epithelium plays an important role in asthma; however, its measurement is challenging. Columnar epithelial cells are often quantified, yet rarely analysed, in induced sputum studies. Objective: We aimed to test whether sputum columnar epithelial cell proportion and count are altered in asthma, and whether they are associated with clinical and inflammatory variables. We aimed to test whether sputum-based measures could provide a relatively non-invasive means through which to monitor airway epithelial activation status. Methods: We examined the relationship of sputum columnar epithelial cells with clinical and inflammatory variables of asthma in a large retrospective cross-sectional cohort (901 participants with asthma and 138 healthy controls). In further studies, we used flow cytometry, microarray, qPCR and ELISA to characterize sputum columnar epithelial cells and their products. Results: Multivariate analysis and generation of 90th centile cut-offs (=11% or =18.1¿×¿104/mL) to identify columnar epithelial cell ¿high¿ asthma revealed a significant relationship between elevated sputum columnar cells and male gender, severe asthma and non-neutrophilic airway inflammation. Flow cytometry showed viable columnar epithelial cells were present in all sputum samples tested. An epithelial gene signature (SCGB3A1, LDLRAD1, FOXJ1, DNALI1, CFAP157, CFAP53) was detected in columnar epithelial cell-high sputum. CLCA1 mRNA and periostin protein, previously identified biomarkers of IL-13-mediated epithelial activation, were elevated in columnar epithelial cell-high sputum samples, but only when accompanied by eosinophilia. Conclusions & clinical relevance: Sputum columnar epithelial cells are related to important clinical and inflammatory variables in asthma. Measurement of epithelial biomarkers in sputum samples could allow non-invasive assessment of altered bronchial epithelium status in asthma.

Purpose: To characterize neutrophils in obstructive airway disease by measuring their surface adhesion molecules and oxidative burst along with characterizing them into different subsets as per their adhesion molecule expression. Patients and methods: Peripheral blood from adults with COPD (n=17), asthma (n=20), and healthy participants (n=19) was examined for expression of CD16, CD62L, CD11b, CD11c, and CD54, and analyzed by flow cytometry. For oxidative burst and CD62L shedding analysis, CD16 and CD62L stained leukocytes were loaded with Dihydrorhodamine-123 (DHR-123) and stimulated with N-Formylmethionine-leucyl-phenylalanine (fMLF). Neutrophil subsets were characterized based on CD16 and CD62L expression. Marker surface expression was recorded on CD16+ neutrophils as median fluorescence intensity (MFI). Results: Neutrophil surface expression of CD62L was significantly reduced in COPD (median (IQR) MFI: 1156 (904, 1365)) compared with asthma (1865 (1157, 2408)) and healthy controls (2079 (1054, 2960)); p=0.028. COPD neutrophils also demonstrated a significant reduction in CD62L expression with and without fMLF stimulation. Asthma participants had a significantly increased proportion and number of CD62Lbright/CD16dim neutrophils (median: 5.4% and 0.14 × 109/L, respectively), in comparison with healthy (3.54% and 0.12 × 109/L, respectively); p<0.017. Conclusion: Reduced CD62L expression suggests blood neutrophils have undergone priming in COPD but not in asthma, which may be the result of systemic inflammation. The increased shedding of CD62L receptor by COPD blood neutrophils suggests a high sensitivity for activation.

Extra-intestinal manifestations (EIM) are common in inflammatory bowel disease (IBD). One such EIM is sub-clinical pulmonary inflammation, which occurs in up to 50% of IBD patients. In animal models of colitis, pulmonary inflammation is driven by neutrophilic infiltrations, primarily in response to the systemic bacteraemia and increased bacterial load in the lungs. Platelet activating factor receptor (PAFR) plays a critical role in regulating pulmonary responses to infection in conditions, such as chronic obstructive pulmonary disease and asthma. We investigated the role of PAFR in pulmonary EIMs of IBD, using dextran sulfate sodium (DSS) and anti-CD40 murine models of colitis. Both models induced neutrophilic inflammation, with increased TNF and IL-1ß levels, bacterial load and PAFR protein expression in mouse lungs. Antagonism of PAFR decreased lung neutrophilia, TNF, and IL-1ß in an NLRP3 inflammasome-dependent manner. Lipopolysaccharide from phosphorylcholine (ChoP)-positive bacteria induced NLRP3 and caspase-1 proteins in human alveolar epithelial cells, however antagonism of PAFR prevented NLRP3 activation by ChoP. Amoxicillin reduced bacterial populations in the lungs and reduced NLRP3 inflammasome protein levels, but did not reduce PAFR. These data suggest a role for PAFR in microbial pattern recognition and NLRP3 inflammasome signaling in the lung.

Inflammatory bowel disease (IBD) is associated with several immune-mediated extraintestinal manifestations. More than half of all IBD patients have some form of respiratory pathology, most commonly neutrophil-mediated diseases, such as bronchiectasis and chronic bronchitis. Using murine models of colitis, we aimed to identify the immune mechanisms driving pulmonary manifestations of IBD. We found increased neutrophil numbers in lung tissue associated with the pulmonary vasculature in both trinitrobenzenesulfonic acid¿ and dextran sulfate sodium¿induced models of colitis. Analysis of systemic inflammation identified that neutrophilia was associated with bacteremia and pyrexia in animal models of colitis. We further identified IL-6 as a systemic mediator of neutrophil recruitment from the bone marrow of dextran sulfate sodium animals. Functional inhibition of IL-6 led to reduced systemic and pulmonary neutrophilia, but it did not attenuate established colitis pathology. These data suggest that systemic bacteremia and pyrexia drive IL-6 secretion, which is a critical driver for pulmonary manifestation of IBD. Targeting IL-6 may reduce neutrophil-associated extraintestinal manifestations in IBD patients.

Biomarkers may be a key foundation for the precision medicine of the future. In this article, we review current knowledge regarding biomarkers in difficult-to-treat asthma and their ability to guide the use of both conventional asthma therapies and novel (targeted) therapies. Biomarkers (as measured by tests including prednisolone and cortisol assays and the fractional exhaled nitric oxide (NO) suppression test) show promise in the assessment and management of non-adherence to inhaled and oral corticosteroids. Multiple markers of type 2 inflammation have been developed, including eosinophils in sputum and blood, exhaled NO, serum IgE and periostin. Although these show potential in guiding the selection of novel interventions for refractory type 2 inflammation in asthma, and in determining if the desired response is being achieved, it is becoming clear that different biomarkers reflect distinct components of the complex type 2 inflammatory pathways. Less progress has been made in identifying biomarkers for use in difficult-to-treat asthma that is not associated with type 2 inflammation. The future is likely to see further biomarker discovery, direct measurements of individual cytokines rather than surrogates of their activity and the increasing use of biomarkers in combination. If the promise of biomarkers is to be fulfilled, they will need to provide useful information that aids clinical decision-making, rather than being ¿just another test¿ for clinicians to order.

Alzheimer's disease is characterized by brain plaques of amyloid beta and by neuronal loss, but it is unclear how amyloid beta causes neuronal loss and how to prevent this loss. We have previously shown that amyloid beta causes neuronal loss by inducing microglia to phagocytose neurons, and here we investigated whether protein kinase Cs and NADPH oxidase were involved in this. The loss of neurons induced by amyloid beta in co-cultures of primary glia and neurons was completely prevented by inhibiting protein kinase Cs with Gö6976 or Gö6983. Directly activating protein kinase Cs with phorbol myristate acetate stimulated microglial phagocytosis, and induced neuronal loss mediated by MFG-E8/vitronectin receptor pathway of microglial phagocytosis. Blocking phagocytosis by MFG-E8 knockout or receptor inhibition left live neurons, indicating microglial phagocytosis was the cause of neuronal death. Phorbol myristate acetate stimulated the microglial NADPH oxidase, and inhibiting the oxidase prevented neuronal loss. A physiological activator of NADPH oxidase, fMLP, also induced neuronal loss dependent on microglia. Amyloid beta-induced neuronal loss was blocked by NADPH oxidase inhibitors, superoxide dismutase or Toll-like receptor function-blocking antibodies. The results indicate that amyloid beta induces microglial phagocytosis of neurons via activating protein kinase Cs and NADPH oxidase, and that activating the kinases or oxidase is sufficient to induce neuronal loss by microglial phagocytosis. Thus inhibiting protein kinase Cs or NADPH oxidase might be beneficial in Alzheimer's disease or other brain pathologies involving inflammatory neuronal loss mediated by microglia.

Protease serine member S31 (Prss31)/transmembrane tryptase/tryptase-¿ is a mast cell (MC)-restricted protease of unknown function that is retained on the outer leaflet of the plasma membrane when MCs are activated. We determined the nucleotide sequences of the Prss31 gene in different mouse strains and then used a Cre/loxP homologous recombination approach to create a novel Prss31 -/- C57BL/6 mouse line. The resulting animals exhibited no obvious developmental abnormality, contained normal numbers of granulated MCs in their tissues, and did not compensate for their loss of the membrane tryptase by increasing their expression of other granule proteases. When Prss31-null MCs were activated with a calcium ionophore or by their high affinity IgE receptors, they degranulated in a pattern similar to that of WT MCs. Prss31-null mice had increased baseline airway reactivity to methacholine but markedly reduced experimental chronic obstructive pulmonary disease and colitis, thereby indicating both beneficial and adverse functional roles for the tryptase. In a cigarette smokeinduced model of chronic obstructive pulmonary disease, WT mice had more pulmonary macrophages, higher histopathology scores, and more fibrosis in their small airways than similarly treated Prss31-null mice. In a dextran sodium sulfate-induced acute colitis model, WT mice lost more weight, had higher histopathology scores, and contained more Cxcl-2 and IL-6 mRNA in their colons than similarly treated Prss31-null mice. The accumulated data raise the possibility that inhibitors of this membrane tryptase may provide additional therapeutic benefit in the treatment of humans with these MC-dependent inflammatory diseases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Introduction: Chronic obstructive pulmonary disease (COPD) is a leading global cause of mortality and chronic morbidity. Inhalation of cigarette smoke is the principal risk factor for development of this disease. COPD is a progressive disease that is typically characterised by chronic pulmonary inflammation, mucus hypersecretion, airway remodelling and emphysema that collectively reduce lung function. There are currently no therapies that effectively halt or reverse disease progression. It is hoped that the development of animal models that develop the hallmark features of COPD, in a short time frame, will aid in the identifying and testing of new therapeutic approaches. Areas covered: The authors review the recent developments in mouse models of chronic cigarette smoke-induced COPD as well as the principal findings. Furthermore, the authors discuss the use of mouse models to understand the pathogenesis and the contribution of infectious exacerbations. They also discuss the investigations of the systemic co-morbidities of COPD (pulmonary hypertension, cachexia and osteoporosis). Expert opinion: Recent advances in the field mark a point where animal models recapitulate the pathologies of COPD patients in a short time frame. They also reveal novel insights into the pathogenesis and potential treatment of this debilitating disease. © 2014 Informa UK, Ltd.