Extracellular Matrix as a Driver of Chronic Lung Diseases.

The extracellular matrix (ECM) is not just a three-dimensional scaffold that provides stable support for all cells in the lungs, but also an important component of chronic fibrotic airway, vascular, and interstitial diseases. It is a bioactive entity that is dynamically modulated during tissue homeostasis and disease, that controls structural and immune cell functions and drug responses, and that can release fragments that have biological activity and that can be used to monitor disease activity. There is a growing recognition of the importance of considering ECM changes in chronic airway, vascular, and interstitial diseases, including 1) compositional changes, 2) structural and organizational changes, and 3) mechanical changes and how these affect disease pathogenesis. As altered ECM biology is an important component of many lung diseases, disease models must incorporate this factor to fully recapitulate disease-driver pathways and to study potential novel therapeutic interventions. Although novel models are evolving that capture some or all of the elements of the altered ECM microenvironment in lung diseases, opportunities exist to more fully understand cell-ECM interactions that will help devise future therapeutic targets to restore function in chronic lung diseases. In this perspective article, we review evolving knowledge about the ECM's role in homeostasis and disease in the lung.

Differential responses of pulmonary vascular cells from PAH patients and controls to TNFα and the effect of the BET inhibitor JQ1.

BACKGROUND: Pulmonary arterial hypertension (PAH) encompasses a group of diseases characterized by raised pulmonary vascular resistance, resulting from vascular remodelling and inflammation. Bromodomain and extra-terminal (BET) proteins are required for the expression of a subset of NF-κB-induced inflammatory genes which can be inhibited by the BET mimic JQ1+. We hypothesised that JQ+ would supress TNFα-driven inflammatory responses in human pulmonary vascular cells from PAH patients. METHODS: Immunohistochemical staining of human peripheral lung tissue (N = 14 PAH and N = 12 non-PAH) was performed for the BET proteins BRD2 and 4. Human pulmonary microvascular endothelial cells (HPMEC) and pulmonary artery smooth muscle cells (HPASMC) from PAH patients (N = 4) and non-PAH controls (N = 4) were stimulated with TNFα in presence or absence of JQ1+ or its inactive isomer JQ1-. IL-6 and -8 mRNA was measured by RT-qPCR and protein levels by ELISA. Chromatin immunoprecipitation analysis was performed using EZ-ChIP™ and NF-κB p65 activation determined using a TransAm kit. MTT assay was used to measure cell viability. RESULTS: Nuclear staining of BRD2 and BRD4 was significantly (p < 0.0001) increased in the lung vascular endothelial and smooth muscle cells from PAH patients compared to controls with normal lung function. TNFα-driven IL-6 release from both HPMECs and HPASMCs was greater in PAH cells than control cells. Levels of CXCL8/IL-8 protein release was higher in PAH HPASMCs than in control cells with similar release observed in HPMECs. TNFα-induced recruitment of activated NF-κB p65 to the IL-6 and CXCL8/IL-8 promoters were similar in both cell types and between subject groups. JQ1+ suppressed TNFα-induced IL-6 and CXCL8/IL-8 release and mRNA expression to a comparable extent in control and PAH HPMECs and HPASMCs. JQ1 had a greater efficacy on IL-6 release in HPMEC and on CXCL8/IL-8 release in HPASMC. CONCLUSION: BET inhibition decreases TNFα driven inflammation in primary pulmonary vascular cells. The anti-inflammatory actions of JQ1 suggests distinct cell-specific regulatory control of these genes. BET proteins could be a target for future therapies for PAH.

IL1RAP expression and the enrichment of IL-33 activation signatures in severe neutrophilic asthma.

BACKGROUND: Interleukin (IL)-33 is an upstream regulator of type 2 (T2) eosinophilic inflammation and has been proposed as a key driver of some asthma phenotypes. OBJECTIVE: To derive gene signatures from in vitro studies of IL-33-stimulated cells and use these to determine IL-33-associated enrichment patterns in asthma. METHODS: Signatures downstream of IL-33 stimulation were derived from our in vitro study of human mast cells and from public datasets of in vitro stimulated human basophils, type 2 innate lymphoid cells (ILC2), regulatory T cells (Treg) and endothelial cells. Gene Set Variation Analysis (GSVA) was used to probe U-BIOPRED and ADEPT sputum transcriptomics to determine enrichment scores (ES) for each signature according to asthma severity, sputum granulocyte status and previously defined molecular phenotypes. RESULTS: IL-33-activated gene signatures were cell-specific with little gene overlap. Individual signatures, however, were associated with similar signalling pathways (TNF, NF-κB, IL-17 and JAK/STAT signalling) and immune cell differentiation pathways (Th17, Th1 and Th2 differentiation). ES for IL-33-activated gene signatures were significantly enriched in asthmatic sputum, particularly in patients with neutrophilic and mixed granulocytic phenotypes. IL-33 mRNA expression was not elevated in asthma whereas the expression of mRNA for IL1RL1, the IL-33 receptor, was up-regulated in the sputum of severe eosinophilic asthma. The mRNA expression for IL1RAP, the IL1RL1 co-receptor, was greatest in severe neutrophilic and mixed granulocytic asthma. CONCLUSIONS: IL-33-activated gene signatures are elevated in neutrophilic and mixed granulocytic asthma corresponding with IL1RAP co-receptor expression. This suggests incorporating T2-low asthma in anti-IL-33 trials.

Association of Differential Mast Cell Activation with Granulocytic Inflammation in Severe Asthma.

Rationale: Mast cells (MCs) play a role in inflammation and both innate and adaptive immunity, but their involvement in severe asthma (SA) remains undefined. Objectives: We investigated the phenotypic characteristics of the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) asthma cohort by applying published MC activation signatures to the sputum cell transcriptome. Methods: Eighty-four participants with SA, 20 with mild/moderate asthma (MMA), and 16 healthy participants without asthma were studied. We calculated enrichment scores (ESs) for nine MC activation signatures by asthma severity, sputum granulocyte status, and three previously defined sputum molecular phenotypes or transcriptome-associated clusters (TACs) 1, 2, and 3 using gene set variation analysis. Measurements and Main Results: MC signatures except unstimulated, repeated FcεR1-stimulated and IFN-γ-stimulated signatures were enriched in SA. A FcεR1-IgE-stimulated and a single-cell signature from asthmatic bronchial biopsies were highly enriched in eosinophilic asthma and in the TAC1 molecular phenotype. Subjects with a high ES for these signatures had elevated sputum amounts of similar genes and pathways. IL-33- and LPS-stimulated MC signatures had greater ES in neutrophilic and mixed granulocytic asthma and in the TAC2 molecular phenotype. These subjects exhibited neutrophil, NF-κB (nuclear factor-κB), and IL-1ß/TNF-α (tumor necrosis factor-α) pathway activation. The IFN-γ-stimulated signature had the greatest ES in TAC2 and TAC3 that was associated with responses to viral infection. Similar results were obtained in an independent ADEPT (Airway Disease Endotyping for Personalized Therapeutics) asthma cohort. Conclusions: Gene signatures of MC activation allow the detection of SA phenotypes and indicate that MCs can be induced to take on distinct transcriptional phenotypes associated with specific clinical phenotypes. IL-33-stimulated MC signature was associated with severe neutrophilic asthma, whereas IgE-activated MC was associated with an eosinophilic phenotype.

Crucial role for lung iron level and regulation in the pathogenesis and severity of asthma.

Accumulating evidence highlights links between iron regulation and respiratory disease. Here, we assessed the relationship between iron levels and regulatory responses in clinical and experimental asthma.We show that cell-free iron levels are reduced in the bronchoalveolar lavage (BAL) supernatant of severe or mild-moderate asthma patients and correlate with lower forced expiratory volume in 1 s (FEV1). Conversely, iron-loaded cell numbers were increased in BAL in these patients and with lower FEV1/forced vital capacity (FVC) ratio. The airway tissue expression of the iron sequestration molecules divalent metal transporter 1 (DMT1) and transferrin receptor 1 (TFR1) are increased in asthma, with TFR1 expression correlating with reduced lung function and increased Type-2 (T2) inflammatory responses in the airways. Furthermore, pulmonary iron levels are increased in a house dust mite (HDM)-induced model of experimental asthma in association with augmented Tfr1 expression in airway tissue, similar to human disease. We show that macrophages are the predominant source of increased Tfr1 and Tfr1+ macrophages have increased Il13 expression. We also show that increased iron levels induce increased pro-inflammatory cytokine and/or extracellular matrix (ECM) responses in human airway smooth muscle (ASM) cells and fibroblasts ex vivo and induce key features of asthma in vivo, including airway hyper-responsiveness (AHR) and fibrosis, and T2 inflammatory responses.Together these complementary clinical and experimental data highlight the importance of altered pulmonary iron levels and regulation in asthma, and the need for a greater focus on the role and potential therapeutic targeting of iron in the pathogenesis and severity of disease.

New drugs under development for COPD.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a lung disease characterized by chronic bronchitis, emphysema, and remodeling. Its prevalence is increasing worldwide; however, there are few effective therapies, and none of the treatments currently available prevent the progression of the disease or target all of the hallmark features. The development and progression of COPD are heterogeneous, which has hampered the development of new therapies. AREAS COVERED: In this review, we cover the emergence of the improvement of existing classes of drugs including glucocorticoids, ß2-adrenoceptor agonists, phosphodiesterase inhibitors, PDE4 selective inhibitors, PDE3/PDE4 inhibitors, protease inhibitors, recombinant α1-antitrypsin and neutrophil elastase inhibitors. We also highlight new compounds that target recently identified mechanisms of COPD, new dual-action muscarinic antagonists, and ß2-agonists, kinase inhibitors, cytokine modifiers, chemokines modifiers, NF-κB inhibitors, senolytics, antioxidants, inhaled antiviral agents, anti-fibrotic compounds, and compounds stimulating lung regeneration. EXPERT OPINION: Given the myriad of potential therapeutic avenues that can be pursued, careful consideration of the phenotypes/endotypes of COPD patients will be important for personalized treatment options in the future, and a full understanding of disease mechanisms in patient subsets will ensure these emerging therapies are targeted appropriately.

The BET Bromodomain Inhibitor I-BET-151 Induces Structural and Functional Alterations of the Heart Mitochondria in Healthy Male Mice and Rats.

The bromodomain and extra-terminal domain family inhibitors (BETi) are a promising new class of anticancer agents. Since numerous anticancer drugs have been correlated to cardiomyopathy, and since BETi can affect non-cancerous tissues, we aimed to investigate in healthy animals any ultrastructural BETi-induced alterations of the heart as compared to skeletal muscle. Male Wistar rats were either treated during 3 weeks with I-BET-151 (2 or 10 mg/kg/day) (W3) or treated for 3 weeks then allowed to recover for another 3 weeks (W6) (3-weeks drug washout). Male C57Bl/6J mice were only treated during 5 days (50 mg/kg/day). We demonstrated the occurrence of ultrastructural alterations and progressive destruction of cardiomyocyte mitochondria after I-BET-151 exposure. Those mitochondrial alterations were cardiac muscle-specific, since the skeletal muscles of exposed animals were similar in ultrastructure presentation to the non-exposed animals. I-BET-151 decreased the respiration rate of heart mitochondria in a dose-dependent manner. At the higher dose, it also decreased mitochondrial mass, as evidenced by reduced right ventricular citrate synthase content. I-BET-151 reduced the right and left ventricular fractional shortening. The concomitant decrease in the velocity-time-integral in both the aorta and the pulmonary artery is also suggestive of an impaired heart function. The possible context-dependent cardiac side effects of these drugs have to be appreciated. Future studies should focus on the basic mechanisms of potential cardiovascular toxicities induced by BETi and strategies to minimize these unexpected complications.

Bromodomain and extra-terminal protein mimic JQ1 decreases inflammation in human vascular endothelial cells: Implications for pulmonary arterial hypertension.

BACKGROUND AND OBJECTIVE: Nuclear factor kappa B (NF-kB)-mediated inflammatory gene expression and vascular endothelial cell proliferation/remodelling are implicated in the pathophysiology of the fatal disease, pulmonary arterial hypertension (PAH). Bromodomain and extra-terminal (BET) proteins are essential for the expression of a subset of NF-kB-induced inflammatory genes. BET mimics including JQ1+ prevent binding of BETs to acetylated histones and down-regulate the expression of selected genes. METHODS: The effects of JQ1+ on the proliferation of primary human pulmonary microvascular endothelial cells (HPMECs) from healthy subjects were measured by bromodeoxyuridine (BrdU) incorporation. Cell cycle progression was assessed by flow cytometry; mRNA and protein levels of cyclin-dependent kinases (CDKs), inhibitors and cytokines were determined by reverse transcription-quantitative PCR (RT-qPCR), Western blotting or ELISA. Histone acetyltransferase (HAT) and deacetylase (HDAC) activities were determined in nuclear extracts from whole lung of PAH and control patients. RESULTS: JQ1+ significantly inhibited IL6 and IL8 (IL6 and CXCL8) mRNA and protein in HPMECs compared with its inactive enantiomer JQ1-. JQ1+ decreased NF-kB p65 recruitment to native IL6 and IL8 promoters. JQ1+ showed a concentration-dependent decrease in HPMEC proliferation compared with JQ1--treated cells. JQ1+ induced G1 cell cycle arrest by increasing the expression of the CDK inhibitors (CDKN) 1A (p21cip ) and CDKN2D (p19INK4D ) and decreasing that of CDK2, CDK4 and CDK6. JQ1+ also inhibited serum-stimulated migration of HPMECs. Finally, HAT activity was significantly increased in the lung of PAH patients. CONCLUSION: Inhibition of BETs in primary HPMECs decreases inflammation and remodelling. BET proteins could be a target for future therapies for PAH.

Glucocorticoids.

The most effective anti-inflammatory drugs used to treat patients with airways disease are topical glucocorticosteroids (GCs). These act on virtually all cells within the airway to suppress airway inflammation or prevent the recruitment of inflammatory cells into the airway. They also have profound effects on airway structural cells to reverse the effects of disease on their function. Glucorticosteroids act via specific receptors-the glucocorticosteroid receptor (GR)-which are a member of the nuclear receptor family. As such, many of the important actions of GCs are to modulate gene transcription through a number of distinct and complementary mechanisms. Targets genes include most inflammatory mediators such as chemokines, cytokines, growth factors and their receptors. GCs delivered by the inhaled route are very effective for most patients and have few systemic side effects. However, in some patients, even high doses of topical or even systemic GCs fail to control their disease. A number of mechanisms relating to inflammation have been reported to be responsible for the failure of these patients to respond correctly to GCs and these provide insight into GC actions within the airways. In these patients, the side-effect profile of GCs prevent continued use of high doses and new drugs are needed for these patients. Targeting the defective pathways associated with GC function in these patients may also reactivate GC responsiveness.

Methemoglobin-induced signaling and chemokine responses in human alveolar epithelial cells.

Diffuse alveolar hemorrhage is characterized by the presence of red blood cells and free hemoglobin in the alveoli and complicates a number of serious medical and surgical lung conditions including the pulmonary vasculitides and acute respiratory distress syndrome. In this study we investigated the hypothesis that exposure of human alveolar epithelial cells to hemoglobin and its breakdown products regulates chemokine release via iron- and oxidant-mediated activation of the transcription factor NF-κB. Methemoglobin alone stimulated the release of IL-8 and MCP-1 from A549 cells via activation of the NF-κB pathway; additionally, IL-8 required ERK activation and MCP-1 required JNK activation. Neither antioxidants nor iron chelators and knockdown of ferritin heavy and light chains affected these responses, indicating that iron and reactive oxygen species are not involved in the response of alveolar epithelial cells to methemoglobin. Incubation of primary cultures of human alveolar type 2 cells with methemoglobin resulted in a similar pattern of chemokine release and signaling pathway activation. In summary, we have shown for the first time that methemoglobin induced chemokine release from human lung epithelial cells independent of iron- and redox-mediated signaling involving the activation of the NF-κB and MAPK pathways. Decompartmentalization of hemoglobin may be a significant proinflammatory stimulus in a variety of lung diseases.

A mechanistic insight into severe COPD: the nose as a surrogate for the airways.

A severe COPD signature in bronchial and nasal epithelial cells reflects reduced tissue repair and ECM regulation https://bit.ly/476S3PJ.

Changes in PD-1- and CTLA-4-bearing blood lymphocytes in ICU COVID-19 patients treated with Favipiravir/Kaletra or Dexamethasone/Remdesivir: a pilot study.

COVID-19, caused by SARS-CoV-2, requires new approaches to control the disease. Programmed cell death protein (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) play important roles in T-cell exhaustion in severe COVID-19. This study evaluated the frequency of whole blood lymphocytes expressing PD-1 and CTLA-4 in COVID-19 patients upon admission to the intensive care unit (ICU) (i.e., severe) or infection ward (i.e., moderate) and after 7 days of antiviral therapy. COVID-19 patients were treated with either favipiravir or Kaletra (FK group, 11 severe and 11 moderate) or dexamethasone plus remdesivir (DR group, 7 severe and 10 moderate) for 7 days in a pilot study. Eight healthy control subjects were also enrolled. The frequency of PD-1+ and CTLA-4+ lymphocytes in whole blood was evaluated by flow cytometry. Patients on DR therapy had shorter hospital stays than those on FK therapy. The frequency of PD-1+ lymphocytes in the FK group at baseline differed between COVID-19 patients and healthy controls, while the frequency of both PD-1+ and CTLA-4+ cells increased significantly 7 days of FK therapy. The response was similar in both moderate and severe patients. In contrast, the frequency of PD-1+ and CTLA-4+ lymphocytes varied significantly between patients and healthy controls before DR treatment. DR therapy enhanced PD-1+ but not the CTLA-4+ frequency of these cells after 7 days. We show that the frequency of PD-1 and CTAL-4-bearing lymphocytes during hospitalization was increased in Iranian ICU COVID-19 patients who received FK treatment, but that the frequency of CTLA-4+ cells was higher at baseline and did not increase in patients who received DR. The effectiveness of DR treatment may reflect differences in T-cell activation or exhaustion status, particularly in CTLA-4-expressing cells.

Characteristics of fine and ultrafine aerosols in the London underground.

Underground railway systems are recognised spaces of increased personal pollution exposure. We studied the number-size distribution and physico-chemical characteristics of ultrafine (PM0.1), fine (PM0.1-2.5) and coarse (PM2.5-10) particles collected on a London underground platform. Particle number concentrations gradually increased throughout the day, with a maximum concentration between 18:00 h and 21:00 h (local time). There was a maximum decrease in mass for the PM2.5, PM2.5-10 and black carbon of 3.9, 4.5 and ~ 21-times, respectively, between operable (OpHrs) and non-operable (N-OpHrs) hours. Average PM10 (52 µg m-3) and PM2.5 (34 µg m-3) concentrations over the full data showed levels above the World Health Organization Air Quality Guidelines. Respiratory deposition doses of particle number and mass concentrations were calculated and found to be two- and four-times higher during OpHrs compared with N-OpHrs, reflecting events such as train arrival/departure during OpHrs. Organic compounds were composed of aromatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) which are known to be harmful to health. Specific ratios of PAHs were identified for underground transport that may reflect an interaction between PAHs and fine particles. Scanning transmission electron microscopy (STEM) chemical maps of fine and ultrafine fractions show they are composed of Fe and O in the form of magnetite and nanosized mixtures of metals including Cr, Al, Ni and Mn. These findings, and the low air change rate (0.17 to 0.46 h-1), highlight the need to improve the ventilation conditions.

Recent evidence from omic analysis for redox signalling and mitochondrial oxidative stress in COPD.

COPD is driven by exogenous and endogenous oxidative stress derived from inhaled cigarette smoke, air pollution and reactive oxygen species from dysregulated mitochondria in activated inflammatory cells within the airway and lung. This is compounded by the loss in antioxidant defences including FOXO and NRF2 and other antioxidant transcription factors together with various key enzymes that attenuate oxidant effects. Oxidative stress enhances inflammation; airway remodelling including fibrosis and emphysema; post-translational protein modifications leading to autoantibody generation; DNA damage and cellular senescence. Recent studies using various omics technologies in the airways, lungs and blood of COPD patients has emphasised the importance of oxidative stress, particularly that derived from dysfunctional mitochondria in COPD and its role in immunity, inflammation, mucosal barrier function and infection. Therapeutic interventions targeting oxidative stress should overcome the deleterious pathologic effects of COPD if targeted to the lung. We require novel, more efficacious antioxidant COPD treatments among which mitochondria-targeted antioxidants and Nrf2 activators are promising.

Lung toxicity of particulates and gaseous pollutants using ex-vivo airway epithelial cell culture systems.

Air pollution consists of a multi-faceted mix of gases and ambient particulate matter (PM) with diverse organic and non-organic chemical components that contribute to increasing morbidity and mortality worldwide. In particular, epidemiological and clinical studies indicate that respiratory health is adversely affected by exposure to air pollution by both causing and worsening (exacerbating) diseases such as chronic obstructive pulmonary disease (COPD), asthma, interstitial pulmonary fibrosis and lung cancer. The molecular mechanisms of air pollution-induced pulmonary toxicity have been evaluated with regards to different types of PM of various sizes and concentrations with single and multiple exposures over different time periods. These data provide a plausible interrelationship between cellular toxicity and the activation of multiple biological processes including proinflammatory responses, oxidative stress, mitochondrial oxidative damage, autophagy, apoptosis, cell genotoxicity, cellular senescence and epithelial-mesenchymal transition. However, these molecular changes have been studied predominantly in cell lines rather than in primary bronchial or nasal cells from healthy subjects or those isolated from patients with airways disease. In addition, they have been conducted under different cell culture conditions and generally in submerged culture rather than the more relevant air-liquid interface culture and with a variety of air pollutant exposure protocols. Cell types may respond differentially to pollution delivered as an aerosol rather than being bathed in media containing agglomerations of particles. As a result, the actual pathophysiological pathways activated by different PMs in primary cells from the airways of healthy and asthmatic subjects remains unclear. This review summarises the literature on the different methodologies utilised in studying the impact of submicron-sized pollutants on cells derived from the respiratory tract with an emphasis on data obtained from primary human cell. We highlight the critical underlying molecular mechanisms that may be important in driving disease processes in response to air pollution in vivo.

Corticosteroid resistance in asthma: Cellular and molecular mechanisms.

Inhaled glucocorticoids (GCs) are drugs widely used as treatment for asthma patients. They prevent the recruitment and activation of lung immune and inflammatory cells and, moreover, have profound effects on airway structural cells to reverse the effects of disease on airway inflammation. GCs bind to a specific receptor, the glucocorticoid receptor (GR), which is a member of the nuclear receptor superfamily and modulates pro- and anti-inflammatory gene transcription through a number of distinct and complementary mechanisms. Targets genes include many pro-inflammatory mediators such as chemokines, cytokines, growth factors and their receptors. Inhaled GCs are very effective for most asthma patients with little, if any, systemic side effects depending upon the dose. However, some patients show poor asthma control even after the administration of high doses of topical or even systemic GCs. Several mechanisms relating to inflammation have been considered to be responsible for the onset of the relative GC resistance observed in these patients. In these patients, the side-effect profile of GCs prevent continued use of high doses and new drugs are needed. Targeting the defective pathways associated with GC function in these patients may also reactivate GC responsiveness.