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BACKGROUND: Mast cells (MC) play a role in inflammation and both innate and adaptive immunity but their involvement in severe asthma (SA) remains undefined. OBJECTIVE: We investigated the phenotypic characteristics of the U-BIOPRED asthma cohort by applying published MC activation signatures to the sputum cell transcriptome. METHODS: 84 SA, 20 mild/moderate (MMA) asthma, and 16 non-asthmatic healthy participants were studied. We calculated enrichment scores (ES) for nine MC activation signatures by asthma severity, sputum granulocyte status and three previously-defined sputum molecular phenotypes or transcriptome-associated clusters (TAC1, 2, 3) using gene-set variation analysis. RESULTS: MC signatures except unstimulated, repeated FcεR1-stimulated and IFNγ-stimulated 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 levels 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, and IL-1β/TNFα pathway activation. The IFNγ-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 asthma cohort. CONCLUSIONS: Gene signatures of MC activation allow the detection of SA phenotypes and indicate that MC can be induced to take on distinct transcriptional phenotypes associated with specific clinical phenotypes. IL-33-stimulated MCs signature was associated with severe neutrophilic asthma while IgE-activated MC with an eosinophilic phenotype.

Introduction/Aim: Clinical and experimental evidence highlights an important role for altered iron metabolism in the pathogenesis and severity of asthma. Elevated iron in airway cells and tissues and increased gene expression of the iron uptake protein, transferrin receptor (TFR)1, correlates with increased Type-2 (T2) cytokine responses, worsened lung function and development of key disease features. Here, we examined the relationship between the T2 cytokine, interleukin (IL)-13 and TFR1 in the pathogenesis of key features of asthma. Methods: Anti-TFR1 (αTFR1) and -IL-13 (αIL-13) was administered intranasally during a murine model of house dust mite (HDM)-induced asthma. The effects of αTFR1 or αIL-13 on inflammatory and TFR1+ cell numbers, collagen deposition and mucus secreting cell (MSC) numbers in the airways, iron metabolism, T2 and pro-inflammatory gene expression or lung function was assessed. The effects of IL-13 on TFR1 and matrix metalloprotease (MMP)9 responses in murine bone marrow derived macrophages (BMDM) was also assessed. Results: TFR1 is increased on macrophages and epithelial cells in HDM-induced asthma. αTFR1 reduces TFR1+ macrophages, but not TFR1+ epithelial cells, in disease. Importantly, αTFR1 reduces inflammation, fibrosis, MSC numbers, airflow obstruction in disease. αTFR1 has no effect on HDM-induced IL-13, but significantly suppresses MMP9, which is implicated in airway remodelling, expression in the lungs. IL-13 increases TFR1 expression in BMDMs and intranasal administration of αIL-13 reduces the numbers of TFR1+ macrophages in HDM-induced asthma. Significantly, the combination of IL-13 and HDM increases MMP9 expression in BMDMs. Conclusion: We show a critical role for IL-13-mediated increases in TFR1 responses in macrophages in driving the pathogenesis of asthma. These findings highlight the potential for the therapeutic manipulation of TFR1 and/or iron metabolism for the treatment of not only asthma but also other respiratory diseases associated with altered iron metabolism such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and cystic fibrosis. Grant Support: University of Newcastle, HMRI.

Background:SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). Circulating soluble angiotensin-converting enzyme (sACE2), the main receptor for SARS-CoV-2, together with components of the renin-angiotensin system promote infection and disease severity. Objective: In this pilot study we followed the time-course of sACE2 levels in relation to systemic cytokines in severe and moderate COVID-19 patients treated with remdesivir/dexamethasone in combination. Methods: Peripheral blood was obtained upon admission from 30 patients (12 with moderate disease and 18 with severe disease) and 14 patients with PCR-confirmed mild COVID-19. Severe and moderate patients were treated with remdesivir (200mg/first day and 100mg/day for the remaining days ) and dexamethasone (100mg/day ). 6 healthy control subjects (HC) were also enrolled. Serum interleukin (IL)-6 and IL-8 and sACE2 levels were measured by ELISA at baseline and during treatment in severe and moderate patients and at baseline in mild and HCs. Results: Baseline sACE2 levels were lower in severe (p=0.0005) and moderate (p=0.0022) patients than in patients with mild COVID-19 and in HC (p=0.0023 and p=0.0012 respectively). Serum sACE2 levels increased in patients with severe disease recovered over time compared with moderate (p=0.0021) and severe (p=0.0411) COVID-19 subjects at baseline. Systemic IL-6 and IL-8 levels were higher in all patient groups compared with HC and were not significantly affected over time or by remdesivir/dexamethasone treatment for 5 days. Conclusion: Serum sACE2 levels increase in severe COVID-19 patients as they recover over time whilst circulating cytokines are unaffected. Future studies should link these results to clinical outcomes. Funding: IMA is financially supported by the Welcome Trust (093080/Z/10/Z), the EPSRC (EP/T003189/1), and the Community Jameel Imperial College COVID-19 Excellence Fund (G26290) and by the UK MRC (MR/T010371/1). SM is supported by EU project 853850. Declaration of Interests: The authors declare that there is no conflict of interest to this article. Ethics Approval Statement: The study was approved by the institutional ethics board of the Masih Daneshvari Hospital (Ethics number SBMU.NRITLD.REC.1399.226).

Neutrophils are main players in the effector phase of the host defense against micro-organisms and have a major role in the innate immune response. Neutrophils show phenotypic heterogeneity and functional flexibility, which highlight their importance in regulation of immune function. However, neutrophils can play a dual role and besides their antimicrobial function, deregulation of neutrophils and their hyperactivity can lead to tissue damage in severe inflammation or trauma. Neutrophils also have an important role in the modulation of the immune system in response to severe injury and trauma. In this review we will provide an overview of the current understanding of neutrophil subpopulations and their function during and post-infection and discuss the possible mechanisms of immune modulation by neutrophils in severe inflammation.

Studies were undertaken to examine any role for the hepcidin/ferroportin axis in proliferative responses of human pulmonary artery smooth muscle cells (hPASMCs). Entirely novel findings have demonstrated the presence of ferroportin in hPASMCs. Hepcidin treatment caused increased proliferation of these cells most likely by binding ferroportin resulting in internalisation and cellular iron retention. Cellular iron content increased with hepcidin treatment. Stabilisation of ferroportin expression and activity via intervention with the therapeutic monoclonal antibody LY2928057 reversed proliferation and cellular iron accumulation. Additionally, IL-6 treatment was found to enhance proliferation and iron accumulation in hPASMCs; intervention with LY2928057 prevented this response. IL-6 was also found to increase hepcidin transcription and release from hPASMCs suggesting a potential autocrine response. Hepcidin or IL-6 mediated iron accumulation contributes to proliferation in hPASMCs; ferroportin mediated cellular iron excretion limits proliferation. Haemoglobin also caused proliferation of hPASMCs; in other novel findings, CD163, the haemoglobin/haptoglobin receptor, was found on these cells and offers a means for cellular uptake of iron via haemoglobin. Il-6 was also found to modulate CD163 on these cells. These data contribute to a better understanding of how disrupted iron homeostasis may induce vascular remodelling, such as in pulmonary arterial hypertension.

Background The acute respiratory distress syndrome (ARDS) is a clinical condition with multiple aetiologies. Previous work has presented two relevant, distinct animal models of ARDS. A murine model of aspiration using acid, in which the lung injury begins to resolve after 48 hours, and another of infection using LPS, that continues to deteriorate. It has been suggested that alveolar epithelial regeneration is implicated in disease progression to different extents between the models. We hypothesised that the wnt and retinoic acid pathways are involved in regeneration and resolution of these murine models of ARDS, and that there is greater activation of these pathways in the aspiration compared to the infection model. Currently, management of ARDS is irrespective of lung injury aetiology, and these mechanisms may prove relevant for pathway modulation for future therapy. Methods Mice were administered either 25 µl intratracheal 0.1 M hydrochloric acid (aspiration model, n=5), 25 µg LPS (infection model, n=6) or sham treated (controls, n=6) and sacrificed at 48 hours. Lung tissue was homogenised. mRNA and protein levels of regenerative pathway genes were quantified, through reverse-transcription polymerase chain reaction and Western blotting. Results Significant upregulation of all 9 regenerative genes studied was seen in the acid model compared to controls. Rac-2 mRNA (wnt pathways) was the only regenerative gene in the LPS-injured mice to show a significant upregulation compared to controls (67.39±25.73 vs 0.32±0.13 ΔCTx1010p

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Long term cigarette smoking is the cause of more than 90% of COPD in Westernized countries. However, only a fraction of chronic heavy smokers develop symptomatic COPD by the age of 80 years. COPD is characterized by an abnormal immune response in the lower airways and its progression is associated with infiltration of the lung by innate and adaptive inflammatory immune cells that form lymphoid follicles. There is growing evidence that both cellular- and antibody-mediated autoimmunity has a fundamental role in the pathogenesis of stable COPD. In particular, carbonyl-modified proteins may help to drive autoimmunity in COPD and to cause the characteristic small airways abnormalities and even contribute to the pathogenesis of pulmonary emphysema. Although direct, indirect, and circumstantial evidence of a role for autoimmunity in stable COPD patients has been identified, no cause-and-effect relationship between autoimmunity and the mechanisms of COPD has been firmly established in man. As such the potential contribution of an autoimmune response to the pathogenesis of COPD exacerbation is still being investigated and represents an area of active research. Many drugs targeting autoimmune responses are already available and the results of controlled clinical trials are awaited with great interest. The potential for measuring specific serum autoantibodies as biomarkers to predict clinical phenotypes or progression of stable COPD is promising.

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.

Haem oxygenase-1 (HO-1) is a highly inducible stress protein that removes haem from cells with the release of biliverdin, carbon monoxide and low-molecular-mass iron (LMrFe). Several antioxidant functions have been ascribed to HO; its induction is considered to be a protective event. However, LMrFe produced during haem catabolism might elicit a pro-oxidant response, with deleterious consequences. We therefore investigated the delicate balance between pro-oxidant and antioxidant events with the use of a microsomal lipid peroxidation (LPO) system. By using microsomal-bound HO in an NADPH-dependent LPO system, we assessed the pro-oxidant nature of the released LMrFe and the antioxidant effect of the released bilirubin. Hb, a biologically relevant substrate for HO, was included with the microsomes to supplement the source of haem iron and to promote LPO. We found significant increases in microsomal LPO, by using the thiobarbituric acid (TBA) test, after incubation with Hb. This Hb-stimulated peroxidation was inhibited by HO inhibitors and by iron chelators, suggesting a HO-driven, iron-dependent mechanism. GLC-MS was employed to measure the specific LPO product 4-hydroxy-2-nonenal and to confirm our TBA test results. A HO inhibitor attenuated an increase in intracellular LMrFe that occurred after treatment of rat pulmonary artery smooth-muscle cells with Hb. Additionally, exogenously added bilirubin at an equimolar concentration to the LMrFe present in both microsomal and liposomal systems was unable to prevent the pro-oxidant effect of the iron. Under certain circumstances HO can act as a pro-oxidant and seems to have a role in stimulating microsomal LPO.

4-Hydroxy-2-nonenal (HNE) an aldehydic peroxidation produce of n-6 fatty acids, is formed predominantly in model systems when redox active transition metal ions, particularly iron, are present. During cardiopulmonary bypass surgery blood is circulated and oxygenated extracorporeally, and periods of tissue ischaemia-reoxygenation are introduced. Both events cause oxidative stress with the generation of reactive forms of oxygen leading to lipid peroxidation. During bypass iron is released often saturating plasma transferrin, and resulting in the appearance of chelatable iron in the plasma. Because such forms of iron can promote HNE formation in vitro, we have studied 12 bypass patients, 5 of whom had plasma iron-overload. Our results show a greater percentage increase in HNE formation during bypass when the plasma transferrin is iron-overloaded compared with matched controls not showing iron-overload.