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Pathological features of both asthma and COPD coexist in some patients and this is termed asthma-COPD overlap (ACO). ACO is heterogeneous and patients exhibit various combinations of asthma and COPD features, making it difficult to characterise the underlying pathogenic mechanisms. There are no controlled studies that define effective therapies for ACO, which arises from the lack of international consensus on the definition and diagnostic criteria for ACO, as well as scantin vitroandin vivodata. There remain unmet needs for experimental models of ACO that accurately recapitulate the hallmark features of ACO in patients. The development and interrogation of such models will identify underlying disease-causing mechanisms, as well as enabling the identification of novel therapeutic targets and providing a platform for assessing new ACO therapies. Here, we review the current understanding of the clinical features of ACO and highlight the approaches that are best suited for developing representative experimental models of ACO.

Background: Smokers undergo frequent and severe respiratory viral infections. We hypothesize that the latter induce pathways eventually leading to COPD. If so, this would allow to identify smokers at increased risk for COPD and therefore their targeted inclusion in smoking cessation and drug intervention programs. Aim: To identify biomarkers associated with predisposition to COPD and to validate these biomarkers in lung samples from COPD patients. Methods: To model heavy smoking, female C57Bl6/J mice were exposed daily to CS or air for 24days. One hour after the last exposure, mice were treated intranasally with PBS or TLR3-ligand PolyIC for 24hrs. Then, BALF was collected and analyzed by 2D-gel electrophoresis coupled with mass-spectrometry (MS). The results were correlated with BAL transcriptome of COPD patients and with lung transcriptome of an independent COPD mouse model. The set of overlapping markers was then confirmed in lung and serum of GOLD stage-III/IV COPD patients. Results: MS showed 41 differentially-expressed proteins in smoke+PolyIC group. The correlation of these proteins with BAL transcriptome of COPD patients showed 7 regulated molecules. One of these molecules, LCN2, was induced in human COPD lung and mouse BALF. The results were confirmed by qRT-PCR, IF and WB. Conclusion: LCN2 was pre-identified in a mouse CS-model with viral trigger. It is abundant in club and ciliated cells and is increased in the bronchiolar epithelium of COPD patients. As a result, it could be affected by CS and viral-trigger since it is localized directly underneath of the apical surface of airway epithelia. To test if LCN2 qualifies as a biomarker predicting COPD it should be assessed in mild COPD and in sputum samples.

Background: Hydrogen sulifde (H2S) is an endogenous anti-inflammatory signalling molecule and the only inorganic electron source for mitochondrial ATP synthesis. Lung H2S levels are depleted in COPD and negatively correlate with smoking pack years. Interventions restoring mitochondrial H2S levels and mitochondrial function could offer a novel COPD treatment option. Objectives: We developed novel molecules (MHD) to target mitochondria by a variety of mechanisms using distinct approaches to deliver minute fluxes of H2S. We examined the effects of MHD at suppressing and reversing CS-induced lung damage, inflammation and lung function in experimental COPD. Methods: Suppression of COPD development-Female C57Bl/6 mice (n=8) were exposed to CS (12 cigarettes,b.i.d.,5 days/week) or air for 8 weeks with mtH (1.0mg/kg,i.p.,q.d). Reversal of COPD-Mice were exposed to CS for 8 weeks followed by either 4 weeks of rest or continued CS exposure with MHD (1.0mg/kg i.n.,q.d). Inflammation (BALF inflammatory cells, H2S and IL-1β) and lung function (forced oscillation and manoeuvre) were assessed. Results: CS exposure depleted lung and mitochondrial H2S levels, induced mitochondrial oxidant production and inflammasome induction. Each MHD significantly suppressed CS-induced IL-1β synthesis, alveolar destruction, fibrosis and improved lung gas exchange (DFCO);each p Conclusion: MHD may be a novel therapeutic approach to treat COPD and related conditions.

Background: Emerging evidence implicates the endoplasmic reticulum-unfolded protein response (ER-UPR) in the pathogenesis of asthma. Type II inflammation in asthmatic airways may induce ER-UPR and contribute to epithelial remodelling however the mechanisms are unknown Aims: To determine if exposure of airway epithelial cells to IL-13 induces ER-UPR and to evaluate the efficacy of ER-UPR inhibitors on IL-13 induced epithelial remodeling Methods: Primary bronchial epithelial cells obtained from healthy (HC) (n=5) and asthmatic (AS) (n=5) subjects were cultured at air-liquid interface (ALI) and treated with IL-13 (10ng/mL) with or without ER-UPR inhibitors 4-PBA (5mM) and TUDCA (0.5mM). ER-UPR was assessed by IHC and immunoblot. Epithelial mucin genes, mucin transcription factors, and pro-fibrotic factors were assessed by qPCR. Secreted mucin assessed by ELISA and IHC Results: IL-13 increased ER-UPR (XBP1s, BiP and EDEM1) in ALIs. 4-PBA reduced IL-13 induced ER-UPR. IL-13 significantly induced MUC5AC gene expression in HC ALIs (73 fold,p=0.02) and AS ALIs (31 fold,p=0.1). Treatment with TUDCA reduced MUC5AC gene expression in HC ALIs by 58% and 48% in AS while 4-PBA reduced it by 99% (p=0.05) in HC ALIs and 98% in AS. Muc5ac ELISA and IHC staining showed significant decrease in secreted mucin with 4-PBA. None of the treatments reduced IL-13 induced STAT6 activation. However, 4-PBA significantly reduced IL-13 induced SPDEF, FOXA3, AGR2 while upregulating FOXA2. Epithelial fibrotic factor periostin was reduced by TUDCA but not 4-PBA while SerpinB2 only reduced by 4-PBA Conclusion: Th2 cytokines upregulate ER-UPR in the airway epithelium. Inhibiting ER-UPR reduces IL-13 induced mucus hypersecretion and epithelial remodeling

Background: Necroptosis is an inflammatory type of regulated cell death, mediated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). COPD involves airway inflammation and loss of alveolar tissue (emphysema), as a consequence of cigarette smoke (CS) inhalation. There is evidence that CS-induced necroptosis contributes to the pathogenesis of COPD. Objectives: We investigated the presence of necroptotic markers in human COPD. To assess the contribution of necroptosis to pulmonary inflammation, airway remodelling and emphysema, we chronically exposed RIPK3- and MLKL-deficient mice to CS. Methods: Protein expression levels of RIPK3 and (phosphorylated) MLKL in lungs of never smokers, smokers without airflow obstruction and COPD patients (GOLD II to IV) were evaluated by immunohistochemistry (IHC) and immunoblotting. Wild-type, RIPK3- and MLKL-deficient mice were exposed to CS for 8 weeks. BALF was collected and total and differential leukocytes were enumerated, and histology of lung tissue was assessed. Results: IHC of human lungs revealed positive staining for RIPK3 and MLKL, primarily in airway epithelial cells. Quantification demonstrated increased expression of MLKL but not RIPK3 in airway epithelium of COPD patients, compared to smokers and never smokers. In addition, phosphorylation of MLKL was increased in lung tissue of patients with severe COPD. RIPK3- and MLKL-deficient mice were protected against inflammation in experimental COPD, and both strains showed protection against CS-induced airway remodelling and emphysema, compared to wild-type controls. Conclusions: We provide evidence that necroptosis contributes to the pathogenesis of chronic disease features of COPD.

Introduction: Severe, steroid-resistant (SSR) asthma is the major unmet need in asthma management and obese asthmatics are more likely to have therapy-resistant disease. Recent studies strongly implicate a role for NLRP3 inflammasome responses in driving obesity-associated AHR, however, these studies did not assess its role in steroid resistance or therapeutic modulation in obesity-associated, SSR asthma. Aims and Objectives: To elucidate the role of NLRP3 inflammasome responses in obesity-associated, SSR asthma. Methods: We developed and characterised a novel murine model of high-fat diet (HFD)-induced, obesity-associated, SSR allergic airways disease (SSR AAD) and assessed the effects of steroid treatment. We also assessed the roles and potential for targeting the NLRP3 inflammasome, and IL-5/IL-13 responses, in our model of HFD-induced, obesity-associated, SSR AAD. Results: HFD administration results in increased weight gain and adiposity; and the development of steroid-resistant AHR. Interestingly, HFD-induced obesity results in increased levels of NLRP3 in the airways and caspase-1 activation in the lung, that are suppressed by treatment with the NLRP3 inflammasome-specific inhibitor, MCC950. Furthermore, combinatorial treatment with anti-IL-5 and anti-IL-13 neutralising antibodies suppresses airway NLRP3 inflammasome responses and steroid-resistant AHR in our model. Conclusions: Our study highlights an unrecognised role for HFD-induced, obesity-associated, NLRP3 inflammasome responses in steroid-resistant AHR. Furthermore, we identify a novel role for IL-5 and IL-13 responses upstream of the NLRP3 inflammasome in the development of obesity-associated, SSR asthma.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of morbidity and death globally. The lack of effective treatments results from an incomplete understanding of the underlying mechanisms driving COPD pathogenesis.Interleukin (IL)-22 has been implicated in airway inflammation and is increased in COPD patients. However, its roles in the pathogenesis of COPD is poorly understood. Here, we investigated the role of IL-22 in human COPD and in cigarette smoke (CS)-induced experimental COPD.IL-22 and IL-22 receptor mRNA expression and protein levels were increased in COPD patients compared to healthy smoking or non-smoking controls. IL-22 and IL-22 receptor levels were increased in the lungs of mice with experimental COPD compared to controls and the cellular source of IL-22 included CD4+ T-helper cells, γδ T-cells, natural killer T-cells and group 3 innate lymphoid cells. CS-induced pulmonary neutrophils were reduced in IL-22-deficient (Il22−/−) mice. CS-induced airway remodelling and emphysema-like alveolar enlargement did not occur in Il22−/− mice. Il22−/− mice had improved lung function in terms of airway resistance, total lung capacity, inspiratory capacity, forced vital capacity and compliance.These data highlight important roles for IL-22 and its receptors in human COPD and CS-induced experimental COPD.

Background: Necroptosis is an inflammatory type of regulated cell death, mediated by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). COPD involves airway inflammation and loss of alveolar tissue (emphysema), as a consequence of cigarette smoke (CS) inhalation. There is evidence that CS-induced necroptosis contributes to the pathogenesis of COPD. Objectives: We investigated the presence of necroptotic markers in human COPD. To assess the contribution of necroptosis to pulmonary inflammation, airway remodelling and emphysema, we chronically exposed RIPK3- and MLKL-deficient mice to CS. Methods: Protein expression levels of RIPK3 and (phosphorylated) MLKL in lungs of never smokers, smokers without airflow obstruction and COPD patients (GOLD II to IV) were evaluated by immunohistochemistry (IHC) and immunoblotting. Wild-type, RIPK3- and MLKL-deficient mice were exposed to CS for 8 weeks. BALF was collected and total and differential leukocytes were enumerated, and histology of lung tissue was assessed. Results: IHC of human lungs revealed positive staining for RIPK3 and MLKL, primarily in airway epithelial cells. Quantification demonstrated increased expression of MLKL but not RIPK3 in airway epithelium of COPD patients, compared to smokers and never smokers. In addition, phosphorylation of MLKL was increased in lung tissue of patients with severe COPD. RIPK3- and MLKL-deficient mice were protected against inflammation in experimental COPD, and both strains showed protection against CS-induced airway remodelling and emphysema, compared to wild-type controls. Conclusions: We provide evidence that necroptosis contributes to the pathogenesis of chronic disease features of COPD.

Severe asthma remains a clinical burden despite the emergence of antibody therapies. Complex disease drivers, which shift between T2, T1 and T17 driven inflammation, termed mixed T cell asthma, likely contribute to ineffective disease control in this population. Here we dissect the mechanisms underlying severe asthma to reveal a potential role (s) for both PI3Kgamma and delta (PI3Kγ and δ) isoform activity in asthmatics with a mixed T cell endotype. A PI3Kγδ gene signature was generated from next generation sequencing of IL-8/GM-CSF-stimulated monocytes where PI3Kγδ signalling was inhibited. This mRNA signature was used to interrogate the Ubiopred database. We observed a strong association between PI3Kγδ pathway activity, severity and mixed eosinophil and neutrophil sputum granulocyte content. To further support a role of PI3Kγδ in a mixed T cell asthma phenotype we demonstrated the following. 1) PI3Kδ-dependent allergen stimulation of mixed T cell activity in grass pollen atopic blood. 2) PI3Kδ-mediated inhibition of IL-5, IFNγ and IL-17 production from asthma patient blood T cells. 3) PI3Kγ mediated signalling was required for activation of asthmatic eosinophil and neutrophil populations. 4) Activity of an inhaled PI3Kγδ dual inhibitor in a rodent in vivo model of steroid resistant allergy/infection on mixed T cell mediator release, granulocyte influx and airways hyperresponsiveness. These data identify a population of mixed T cell asthma patients with increased PI3Kγδ activity and suggest a therapeutic potential of an inhaled PI3K dual inhibitor.

Introduction: Steroid-resistant (SR) asthma represents a significant clinical and economic burden. The mechanisms which drive SR asthma are unknown, but respiratory infections have been implicated in the development of disease. Infections increase oxidative stress in the lung and SR asthma is associated with defective antioxidant responses and increased oxidative stress. Hypotheses: Respiratory infections induce changes in the asthmatic airway that increase oxidative stress and contribute to dysregulated antioxidant responses which play a crucial role in the development of SR asthma. Aim: To determine the effects of antioxidant therapy on oxidative stress, inflammation and airways hyper-responsiveness (AHR) in a murine model of Chlamydia -induced SR asthma. Methods: Oxidative stress was measured in our established model of Chlamydia -induced SR allergic airways disease (AAD) using 3-nitrotyrosine and 8-isoprostane assays. The effects of treatment with vitamin E or an NRF2 activator on oxidative stress, inflammation and AHR in SRAAD were assessed compared to dexamethasone (DEX). Results: We show that oxidative stress responses are increased in Chlamydia -induced SRAAD compared to controls with steroid-sensitive AAD. Oxidative stress responses in SRAAD are not affected by DEX. Vitamin E treatment alone reduces oxidative stress and, when combined with DEX, suppresses oxidative stress, inflammation and AHR, in SRAAD. Treatment with the NRF2 activator suppresses all features of SRAAD in the absence of DEX. Conclusions: SRAAD is associated with increased oxidative stress that are resistant to steroids. Our findings suggest that antioxidant therapy may be appropriate for SR asthma especially when combined with steroid therapy.

Introduction: Steroid-insensitive (SI) asthma is of considerable clinical and economic significance and improved therapies are urgently required, however, the development of therapies has been hampered by a lack of understanding of the processes that underpin disease. Aim: To develop experimental models of SI asthma that represent different subtypes of clinical disease and use these models to elucidate universal drivers of SI asthma. Methods: Mouse models of Chlamydia , Haemophilus , influenza and respiratory syncytial virus (RSV) lung infections were superimposed with a model of steroid-sensitive ovalbumin (Ova)-induced allergic airways disease (AAD). The effects of infection on AAD and response to dexamethasone (DEX) treatment were examined. Results: We show that Chlamydia and Haemophilus infections drive neutrophil-dominated inflammation, while influenza and RSV infections drive eosinophil-dominated inflammation, in AAD. DEX does not suppress inflammation or airways hyper-responsiveness in all four models. These models of SIAAD represent neutrophil and eosinophil-enriched subtypes of SI asthma. Using gene expression and microRNA (miR) array analyses of these models we identified several factors that are universally dysregulated in SIAAD. Significantly, we show that suppression of miR-21 is effective for restoring steroid sensitivity in all four models. Conclusion: We have developed unique models of SI asthma and used these models to identify factors that are universally dysregulated in SI disease. These factors may represent novel targets for therapies that are broadly effective for the treatment of different subtypes of SI asthma in humans.

Introduction: Excessive NLRP3 inflammasome and concomitant IL-1β responses are implicated in many inflammatory diseases. However, the direct contributions to pathogenesis, mechanisms involved and potential for therapeutic targeting remain poorly understood. In the lung, NLRP3 inflammasome and IL-1β are associated with emphysema, infections and steroid-resistant (SR) asthma, which is the major unmet clinical need in asthma management. Aim: To investigate the role of the NLRP3 inflammasome and IL-1β in SR asthma. Methods: We developed mouse models of Chlamydia , and Haemophilus , respiratory infection-mediated, ovalbumin-induced SR allergic airways disease (SRAAD). These models share the hallmark features of human disease, including elevated neutrophils in the airways, NLRP3 inflammasome and IL-1β responses. The roles and potential for targeting of NLRP3 inflammasome, caspase-(CASP)1, and IL-1β responses in the lung in SRAAD were examined using a highly-selective NLRP3 inhibitor, MCC950, the specific CASP1 inhibitor, Ac-YVAD-cho, and neutralising anti-IL-1β antibody, α-IL-1β, respectively. Results: We show that Chlamydia and Haemophilus infections increase NLRP3, CASP1, IL-1β and T H 1/17 responses that drive steroid-resistant neutrophilic inflammation and airways hyper-responsiveness in SRAAD. Neutrophilic airway inflammation and severity of human SR asthma correlated with IL-1β and NLRP3 expression. Treatment with α-IL-1β , Ac-YVAD-cho, and MCC950 suppressed IL-1β responses and the important steroid-resistant features of disease. Conclusions: NLRP3 inflammasome responses may drive SR asthma and be therapeutically targeted in this and other NLRP3-mediated diseases.