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Background Upper respiratory tract infections (URTIs) are important triggers for asthma exacerbations. We hypothesized that inhalation of the anti‐viral cytokine, interferon (IFN)‐β, during URTI, could prevent these exacerbations. Objective To evaluate the efficacy of on‐demand inhaled IFN‐β1a (AZD9412) to prevent severe asthma exacerbations following symptomatic URTI. Methods This was a randomized, double‐blind, placebo‐controlled trial in which patients with severe asthma (GINA 4‐5; n = 121) reporting URTI symptoms were randomized to 14 days of once‐daily nebulized AZD9412 or placebo. The primary endpoint was severe exacerbations during treatment. Secondary endpoints included 6‐item asthma control questionnaire (ACQ‐6) and lung function. Exploratory biomarkers included IFN‐response markers in serum and sputum, blood leucocyte counts and serum inflammatory cytokines. Results Following a pre‐planned interim analysis, the trial was terminated early due to an unexpectedly low exacerbation rate. Asthma worsenings were generally mild and tended to peak at randomization, possibly contributing to the lack of benefit of AZD9412 on other asthma endpoints. Numerically, AZD9412 did not reduce severe exacerbation rate, ACQ‐6, asthma symptom scores or reliever medication use. AZD9412 improved lung function (morning peak expiratory flow; mPEF) by 19.7 L/min. Exploratory post hoc analyses indicated a greater mPEF improvement by AZD9412 in patients with high blood eosinophils (>0.3 × 109/L) at screening and low serum interleukin‐18 relative change at pre‐treatment baseline. Pharmacodynamic effect of AZD9412 was confirmed using IFN‐response markers. Conclusions & Clinical Relevance Colds did not have the impact on asthma patients that was expected and, due to the low exacerbation rate, the trial was stopped early. On‐demand AZD9412 treatment did not numerically reduce the number of exacerbations, but did attenuate URTI‐induced worsening of mPEF. Severe asthma patients with high blood eosinophils or low serum interleukin‐18 response are potential subgroups for further investigation of inhaled IFN‐β1a.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection carries a substantial risk of severe and prolonged illness; treatment options are currently limited. We assessed the efficacy and safety of inhaled nebulised interferon beta-1a (SNG001) for the treatment of patients admitted to hospital with COVID-19.MethodsWe did a randomised, double-blind, placebo-controlled, phase 2 pilot trial at nine UK sites. Adults aged 18 years or older and admitted to hospital with COVID-19 symptoms, with a positive RT-PCR or point-of-care test, or both, were randomly assigned (1:1) to receive SNG001 (6 MIU) or placebo by inhalation via a mouthpiece daily for 14 days. The primary outcome was the change in clinical condition on the WHO Ordinal Scale for Clinical Improvement (OSCI) during the dosing period in the intention-to-treat population (all randomised patients who received at least one dose of the study drug). The OSCI is a 9-point scale, where 0 corresponds to no infection and 8 corresponds to death. Multiple analyses were done to identify the most suitable statistical method for future clinical trials. Safety was assessed by monitoring adverse events for 28 days. This trial is registered with Clinicaltrialsregister.eu (2020-001023-14) and ClinicalTrials.gov (NCT04385095); the pilot trial of inpatients with COVID-19 is now completed.FindingsBetween March 30 and May 30, 2020, 101 patients were randomly assigned to SNG001 (n=50) or placebo (n=51). 48 received SNG001 and 50 received placebo and were included in the intention-to-treat population. 66 (67%) patients required oxygen supplementation at baseline: 29 in the placebo group and 37 in the SNG001 group. Patients receiving SNG001 had greater odds of improvement on the OSCI scale (odds ratio 2·32 [95% CI 1·07-5·04]; p=0·033) on day 15 or 16 and were more likely than those receiving placebo to recover to an OSCI score of 1 (no limitation of activities) during treatment (hazard ratio 2·19 [95% CI 1·03-4·69]; p=0·043). SNG001 was well tolerated. The most frequently reported treatment-emergent adverse event was headache (seven [15%] patients in the SNG001 group and five [10%] in the placebo group). There were three deaths in the placebo group and none in the SNG001 group.InterpretationPatients who received SNG001 had greater odds of improvement and recovered more rapidly from SARS-CoV-2 infection than patients who received placebo, providing a strong rationale for further trials.FundingSynairgen Research.

Sarcoidosis is a multisystem disease of unknown aetiology. Up to 90% of sarcoidosis patients have lung involvement including pulmonary fibrosis. This leads to impaired quality of life and sometimes mortality. To better understand the nature of the lung matrix we generated novel 3D multicellular foci from individual patient cells. Primary lung fibroblasts were isolated from explant tissue from patients with sarcoidosis. These were then grown in optimal conditions to promote 3D multicellular foci formation for a total of six weeks, treated +/- transforming growth factor-beta-1 (TGFβ1). Alpha smooth muscle actin (aSMA) and collagen mRNA expression were assess­ed by RTqPCR and gross mechanical stiffness of the resulting multicellular foci was assessed by parallel plate compression testing. Total collagen was assessed by hydroxyproline assay (ELISA), pyridinoline (PYD) concentrations were measured by ELISA and LC-MS to assess collagen cross-linking. Treatment of sarcoidosis fibroblasts with 2.5ng/mL TGFβ1 caused a 6-fold increase in collagen-1 (p TGFβ1 increases fibrotic markers in sarcoidosis fibroblasts cultured in this long-term cell culture model.

Background: Interferon beta (IFN-β) plays a key role in host defence against viruses, triggering the expression of interferon stimulated genes (ISG) which restrict viral replication and promote antiviral immune responses. Inhaled IFN-β is being developed as a treatment for viral exacerbations of lung disease. In previous studies, treatment with inhaled IFN-β boosted lung antiviral defences (sputum cell ISG expression) and improved lung function and symptoms in ‘difficult-to-treat’ asthmatics with a cold or flu infection (Djukanovic AJRCCM 2014 190(2):145-54). SG015 (NCT03570359) is an ongoing study in COPD patients, without (Part 1) and with (Part 2) a confirmed respiratory virus infection, assessing antiviral biomarker responses and clinical effects of inhaled IFN-β compared to placebo. Results from Part 1 are presented. Methods: 10 patients received three daily doses of IFN-β (6MIU) or placebo (8:2 ratio) via a nebuliser. Sputum was collected prior to dosing (baseline), 24hrs post first and third dose and 4-6 days after their third dose. Sputum cell gene expression (Mx1, OAS1, IFIT2, GBP1 and CXCL10) was determined by RT-qPCR. Results: Inhaled IFN-β was well tolerated. All antiviral biomarkers were significantly upregulated 24hrs post dose compared to baseline (p Conclusions: Antiviral biomarkers were significantly upregulated in the lungs of COPD patients following the administration of inhaled IFN-β, supporting the proposed mechanism-of-action as a treatment for virus-induced exacerbations.

Background: Increased tissue stiffness is a consequence and a driver of fibrosis. Tissue stiffness is modulated by the lysyl oxidase (LOX) family of enzymes, which cross-link collagen and elastin fibres. Increased expression of LOX-like-2 (LOXL2) is associated with the development of fibrosis and therefore a therapeutic target. We have profiled novel orally bioavailable LOXL2-selective small molecule inhibitors in in vitro and in vivo models of lung fibrosis. Methods: In the in vitro model, lung fibroblasts from IPF patients were cultured under optimised conditions for mature collagen matrix deposition. Following TGF-β1 treatment, multicellular foci formed which were histochemically similar in organization to fibroblastic foci in vivo (Jones et al., AJRCCM 191;2015:A4912). Transient overexpression of active TGF-β1 by adenovector gene transfer in rat lungs results in severe progressive fibrosis (Sime et al JCI 1997;100:768–776). The effects of treatment with a LOXL2-selective inhibitor (days 2-28) on histology and lung function were assessed at day 28. Results: Treatment with LOXL2-selective inhibitors dose-dependently reduced cross-link formation, altered the organisation of collagen matrix and reduced matrix stiffness (assessed by parallel plate compression) in the in-vitro model. In vivo, treatment at 15 or 30 mg/kg significantly lowered lung elastance compared to vehicle control from 2.2 (0.39) to 1.7 (0.31) or 1.6 (0.31) respectively (mean (SD) cmH2O/ml; p Conclusions The results suggest that inhibition of LOXL2 using these novel inhibitors has the potential to improve lung function in patients with lung fibrosis by reducing tissue stiffness.

To the Editor : The majority of asthma exacerbations are associated with respiratory virus infections, mostly rhinoviruses (RVs) [1], due to enhanced inflammation in the airways [2]. These occur despite symptom control with inhaled corticosteroids (ICS) [3]. Experimental RV infection is a valuable tool for studying virus-induced exacerbations [2, 4], but has, to date, involved only corticosteroid-naive asthmatics. We have, therefore, modified a validated infection protocol [4] to inoculate 11 subjects whose asthma was well controlled with ICS. As this was the first experimental infection in patients at risk of severe exacerbations, a cautious study design was implemented. All subjects were followed-up twice daily by SMS text messages during the study. We used RV16, a strain used safely in previous studies, which replicates in vitro to a similar extent but induces less inflammation and cell death than other strains [5]. We also chose a 10-fold lower inoculation dose of the same stock used in previous studies [6]. The design allowed for dose escalation if necessary (this proved to be unnecessary as all subjects developed cold symptoms at this dose). As a final precaution, the delivery device generated particles of 30–100 μm, restricting delivery to the nose (aerosols ≥16 μm are deposited in the upper respiratory tract (URT) [7]), thus closely mimicking natural infection, i.e. limiting direct lung exposure during inoculation. Symptoms of URT infection, asthma, and measurements of lower respiratory tract (LRT) function were recorded post-inoculation. Infection was confirmed by quantitative (q)PCR for RV16 in nasal lavage and sputum and by determining serum anti-RV16 titres. At least a ≥four-fold increase in titres in convalescent serum or shedding of RV16 in the airways was evidence of successful infection. We also studied innate immune responses …

Introduction: The lung tissue of patients with idiopathic pulmonary fibrosis (IPF) is characterised by dense collections of myofibroblasts and extracellular matrix (ECM) termed ‘fibroblastic foci’. Using a novel in vitro model of fibroblastic foci (Jones et al., AJRCCM 191;2015:A4912) we have characterised the formation of lysyl-oxidase (LOX) mediated collagen cross-links …

Introduction: A feature of scarring in IPF is increased collagen deposition and stiffening by lysyl oxidase (LOX)-family mediated cross-links. LOXL2 is an attractive target for treatment since expression is increased in IPF (Barry-Hamilton et al. Nat Med 2010;16,9:1009-1018) and increased serum LOXL2 is associated with more rapid disease progression (Chien et al. ERJ 2014;43:1430-1438). Compounds A and B are novel LOXL2 selective inhibitors, with 125- and 40-fold selectivity for LOXL2 over LOX. Aim: To study the effects of compounds A and B on LOX-mediated cross-links in an in-vitro model of fibroblastic foci using IPF-derived human peripheral lung fibroblasts (IHLF). Method: IHLF were obtained from diagnostic biopsies from confirmed cases of IPF. Cells were seeded onto transwells under conditions appropriate for mature collagen matrix generation ± 1mM BAPN, or compound A or B at 0.001-10nM. After 1 week cultures were treated with TGF-β to induce transformation and contraction of the cultures to spheres. After 6 weeks cultures were harvested, chemically reduced to stabilise bi-valent cross-links, and acid hydrolysed. Pyridinoline (Pyd) cross-links were assessed by ELISA. Results: Pyd cross-links increased from 24.6 to 108ng per mg total protein (1.5 to 3.1ng Pyd per µg Hyp) from 2 to 6 weeks. Pyd formation was inhibited in a dose-dependent manner by both compound A and B, by 68% and 73% respectively at 1nM, and >90% at 10nM, after 6 weeks of culture. Conclusions: The LOXL2 selective compounds inhibit formation of Pyd cross-links at concentrations below the cell-free IC50 for LOX in an in-vitro IHLF model of fibroblastic foci.

BACKGROUND: Nonasthmatic eosinophilic bronchitis is a condition characterized by the presence of eosinophilic airway inflammation in the absence of airflow obstruction or airway hyperresponsiveness. In asthma, the T H 2-type cytokine IL-13 has been implicated in the development of airway inflammation and hyperresponsiveness. Whether the expression of IL-13 is different between these 2 conditions is unknown. OBJECTIVE: We sought to investigate whether IL-13 expression is increased in asthma compared with eosinophilic bronchitis. METHODS: Sputum samples from subjects with mild asthma (n = 30) and eosinophilic bronchitis (n = 15) and normal controls (n = 16) were dialyzed, and IL-13 concentration was measured by ELISA. In a subgroup of these patients, IL-13 protein expression in bronchial biopsies was assessed by immunohistochemistry. RESULTS: The concentration of sputum IL-13 was higher in patients with mild asthma than in normal controls ( P = .03) and in patients with eosinophilic bronchitis ( P = .03). The median (interquartile range) number of IL-13 + cells/mm 2 submucosa was significantly higher in asthma 4 (8) than eosinophilic bronchitis 1.7 (1.9) and normal controls 0.5 (1.1; P = .004). Eighty-three percent of the cells expressing IL-13 in the submucosa were eosinophils, and 8% were mast cells. The median (interquartile range) proportion of eosinophils that expressed IL-13 was higher in the subjects with asthma, 16 (10)%, than those with eosinophilic bronchitis, 7 (3)% ( P = .02). CONCLUSION: The increased expression of IL-13 in asthma compared with eosinophilic bronchitis supports the concept that IL-13 may play a critical role in the pathophysiology of asthma.

BACKGROUND: The importance of IL-13 in the asthma paradigm is supported by increased expression in human subjects, particularly in patients with mild-to-moderate asthma. However, the role of IL-13 in severe asthma needs to be further defined. OBJECTIVE: We sought to assess IL-13 expression in sputum and bronchial biopsy specimens from subjects with mild-to-severe asthma. METHODS: Sputum IL-13 concentrations were measured in 32 control subjects, 34 subjects with mild asthma, 21 subjects with moderate asthma, and 26 subjects with severe asthma. Enumeration of mast cells, eosinophils, and IL-13+ cells in the bronchial submucosa and airway smooth muscle (ASM) bundle was performed in 7 control subjects, 14 subjects with mild asthma, 7 subjects with moderate asthma, and 7 subjects with severe asthma. RESULTS: The proportion of subjects with measurable IL-13 in the sputum was increased in the mild asthma group (15/34) and severe asthma group (10/26) compared with that seen in the control group (4/32; P = .004). IL-13+ cells were increased within the submucosa in all asthma severity groups compared with control subjects (P = .006). The number of IL-13+ cells were increased within the ASM bundle in the severe asthma group compared with that seen in the other groups (P < .05). Asthma control questionnaire scores positively correlated with sputum IL-13 concentrations (R(s) = 0.35, P = .04) and mast cells in the ASM bundle (R(s) = 0.7, P = .007). IL-13+ cells within the submucosa and ASM correlated with sputum eosinophilia (R(s) = 0.4, P < or = .05). CONCLUSIONS: IL-13 overexpression in sputum and bronchial biopsy specimens is a feature of severe asthma.

Background and Purpose For antibody therapies against receptor targets, in vivo outcomes can be difficult to predict because of target-mediated clearance or antigen ‘sink’ effects. The purpose of this work was to engineer an antibody to the GM-CSF receptor α (GM-CSFRα) with pharmacological properties optimized for chronic, s.c. treatment of rheumatoid arthritis (RA) patients. Experimental Approach We used an in silico model of receptor occupancy to guide the target affinity and a combinatorial phage display approach for affinity maturation. Mechanism of action and internalization assays were performed on the optimized antibody in vitro before refining the modelling predictions of the eventual dosing in man. Finally, in vivo pharmacology studies in cynomolgus monkeys were carried out to inform the predictions and support future clinical development. Key Results Antibody potency was improved 8600-fold, and the target affinity was reached. The refined model predicted pharmacodynamic effects at doses as low as 1 mg kg−1 and a study in cynomolgus monkeys confirmed in vivo efficacy at 1 mg kg−1 dosing. Conclusions and Implications This rational approach to antibody drug discovery enabled the isolation of a potent molecule compatible with chronic, s.c. self-administration by RA patients. We believe this general approach enables the development of optimal biopharmaceuticals.

The airway epithelium forms a physical, chemical and immunological barrier against inhaled environmental substances. In asthma, these barrier properties are thought to be abnormal. In this study, we analysed the effect of grass pollen on the physical and immunological barrier properties of differentiated human primary bronchial epithelial cells. Following exposure to Timothy grass (Phleum pratense) pollen extract, the integrity of the physical barrier was not impaired as monitored by measuring the transepithelial resistance and immunofluorescence staining of tight junction proteins. In contrast, pollen exposure affected the immunological barrier properties by modulating vectorial mediator release. CXC chemokine ligand (CXCL)8/interleukin (IL)-8 showed the greatest increase in response to pollen exposure with preferential release to the apical compartment. Inhibition of the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways selectively blocked apical CXCL8/IL-8 release via a post-transcriptional mechanism. Apical release of CC chemokine ligand (CCL)20/macrophage inflammatory protein-3α, CCL22/monocyte-derived chemokine and tumour necrosis factor-α was significantly increased only in severe asthma cultures, while CCL11/eotaxin-1 and CXCL10/interferon-γ-induced protein-10 were reduced in nonasthmatic cultures. The bronchial epithelial barrier modulates polarised release of mediators in response to pollen without direct effects on its physical barrier properties. The differential response of cells from normal and asthmatic donors suggests the potential for the bronchial epithelium to promote immune dysfunction in asthma.

BACKGROUND AND PURPOSE IL-13 is a pleiotropic Th2 cytokine considered likely to play a pivotal role in asthma. Here we describe the preclinical in vitro and in vivo characterization of CAT-354, an IL-13-neutralizing IgG4 monoclonal antibody (mAb), currently in clinical development. EXPERIMENTAL APPROACH In vitro the potency, specificity and species selectivity of CAT-354 was assayed in TF-1 cells, human umbilical vein endothelial cells and HDLM-2 cells. The ability of CAT-354 to modulate disease-relevant mechanisms was tested in human cells measuring bronchial smooth muscle calcium flux induced by histamine, eotaxin generation by normal lung fibroblasts, CD23 upregulation in peripheral blood mononuclear cells and IgE production by B cells. In vivo CAT-354 was tested on human IL-13-induced air pouch inflammation in mice, ovalbumin-sensitization and challenge in IL-13 humanized mice and antigen challenge in cynomolgus monkeys. KEY RESULTS CAT-354 has a 165 pM affinity for human IL-13 and functionally neutralized human, human variant associated with asthma and atopy (R130Q) and cynomolgus monkey, but not mouse, IL-13. CAT-354 did not neutralize human IL-4. In vitro CAT-354 functionally inhibited IL-13-induced eotaxin production, an analogue of smooth muscle airways hyperresponsiveness, CD23 upregulation and IgE production. In vivo in humanized mouse and cynomolgus monkey antigen challenge models CAT-354 inhibited airways hyperresponsiveness and bronchoalveolar lavage eosinophilia. CONCLUSIONS AND IMPLICATIONS CAT-354 is a potent and selective IL-13-neutralizing IgG4 mAb. The preclinical data presented here support the trialling of this mAb in patients with moderate to severe uncontrolled asthma.

Background: The Th2 cytokine interleukin (IL)-13 is implicated in the development of various allergic diseases including asthma. The IL-13 receptor, IL-13Rα1, is expressed on most leukocytes, except T-cells. Evidence to support IL-13Rα1 expression on mast cells is limited. Methods: We investigated: (i) IL-13Rα1 expression by human lung mast cells (HLMC); (ii) the number of IL-13Rα1+ bronchial submucosal mast cells in subjects with asthma and normal controls and (iii) the effect of IL-13 priming on HLMC expression of high-affinity IgE receptor (FcɛRI), stem cell factor receptor (CD117), histamine release, proliferation, and survival. Results: Human lung mast cell expressed IL-13Rα1 mRNA. IL-13Rα1 was highly expressed on the surface HLMC (82 ± 9%). Bronchial submucosal mast cell IL-13Rα1 expression was higher in asthmatics (86 ± 2%) than normal controls (78 ± 2%; P = 0.015). IL-13 priming for 30 min did not increase HLMC histamine release, in the presence or absence of SCF or in response to IgE/anti-IgE activation. IL-13 priming for 5 days upregulated HLMC FcɛRI expression (22% increase in fluorescent intensity; P = 0.003), increased histamine release following IgE/anti-IgE activation by 56% (P = 0.03) and increased proliferation by 50% (P = 0.003) without affecting cell survival or CD117 expression. The IL-13 specific neutralizing antibody CAT-354 inhibited all IL-13 mediated effects. Conclusion: Human lung mast cell express IL-13Rα1 and activation by IL-13 for 5 days increased FcɛRI expression and proliferation. Histamine release was not affected by short-term priming with IL-13, but was upregulated by priming for 5 days suggesting that this effect was mediated by the increased FcɛRI expression. These data support the view that targeting IL-13 may be beneficial in the treatment of asthma.

Rationale: Ex vivo, bronchial epithelial cells from people with asthma are more susceptible to rhinovirus infection caused by deficient induction of the antiviral protein, IFN-β. Exogenous IFN-β restores antiviral activity. Objectives: To compare the efficacy and safety of inhaled IFN-β with placebo administered to people with asthma after onset of cold symptoms to prevent or attenuate asthma symptoms caused by respiratory viruses. Methods: A total of 147 people with asthma on inhaled corticosteroids (British Thoracic Society Steps 2–5), with a history of virus-associated exacerbations, were randomized to 14-day treatment with inhaled IFN-β (n = 72) or placebo (n = 75) within 24 hours of developing cold symptoms and were assessed clinically, with relevant samples collected to assess virus infection and antiviral responses. Measurements and Main Results: A total of 91% of randomized patients developed a defined cold. In this modified intention-to-treat population, asthma symptoms did not get clinically significantly worse (mean change in six-item Asthma Control Questionnaire

The critical role played by IgE in allergic asthma is well-documented and clinically precedented, but some patients in whom IgE neutralization may still offer clinical benefit are excluded from treatment with the existing anti-IgE therapy, omalizumab, due to high total IgE levels or body mass. In this study, we sought to generate a novel high affinity anti-IgE antibody (MEDI4212) with potential to treat a broad severe asthma patient population. Analysis of body mass, total and allergen-specific IgE levels in a cohort of severe asthmatics was used to support the rationale for development of a high affinity IgE-targeted antibody therapeutic. Phage display technology was used to generate a human IgG1 lead antibody, MEDI4212, which was characterized in vitro using binding, signaling and functional assay systems. Protein crystallography was used to determine the details of the interaction between MEDI4212 and IgE. MEDI4212 bound human IgE with an affinity of 1.95 pM and was shown to target critical residues in the IgE Ce3 domain critical for interaction with FceRI. MEDI4212 potently inhibited responses through FceRI and also prevented the binding of IgE to CD23. When used ex vivo at identical concentration, MEDI4212 depleted free-IgE from human sera to levels ~1 log lower than omalizumab. Our results thus indicate that MEDI4212 is a novel, high affinity antibody that binds specifically to IgE and prevents IgE binding to its receptors. MEDI4212 effectively depleted free-IgE from human sera ex vivo to a level (1 IU/mL) anticipated to provide optimal IgE suppression in severe asthma patients.

Asthma is a complex disease involving gene and environment interactions. Although atopy is a strong predisposing risk factor for asthma, local tissue susceptibilities are required for disease expression. The bronchial epithelium forms the interface with the external environment and is pivotally involved in controlling tissue homeostasis through provision of a physical barrier controlled by tight junction (TJ) complexes.To explain the link between environment exposures and airway vulnerability, we hypothesized that epithelial TJs are abnormal in asthma, leading to increased susceptibility to environmental agents.Localization of TJs in bronchial biopsies and differentiated epithelial cultures was assessed by electron microscopy or immunostaining. Baseline permeability and the effect of cigarette smoke and growth factor were assessed by measurement of transepithelial electrical resistance and passage of fluorescently labeled dextrans.By using immunostaining, we found that bronchial biopsies from asthmatic subjects displayed patchy disruption of TJs. In differentiated bronchial epithelial cultures, TJ formation and transepithelial electrical resistance were significantly lower (P.05) in cultures from asthmatic donors (n = 43) than from normal controls (n = 40) and inversely correlated with macromolecular permeability. Cultures from asthmatic donors were also more sensitive to disruption by cigarette smoke extract. Epidermal growth factor enhanced basal TJ formation in cultures from asthmatic subjects (P.01) and protected against cigarette smoke-induced barrier disruption (P.01).Our results show that the bronchial epithelial barrier in asthma is compromised. This defect may facilitate the passage of allergens and other agents into the airway tissue, leading to immune activation and may thus contribute to the end organ expression of asthma.

HuFceRI transfected RBL or LAD2 cells were incubated with IgE ± MEDI4212. Bound IgE was cross-linked using a commercial anti-IgE and cell activation detected using calcium signalling or β-hexosaminidase release. Effect of MEDI4212 on IgE/CD23 interaction was assessed by incubation of IL-4 stimulated IM9 cells with IgE ± MEDI4212 and bound IgE detected by flow cytometry. Effects on IgE production in human B cells (differentiated with APC + IL-4, ± MEDI4212) was evaluated by flow cytometry. Serum free-IgE was measured using ELISA.

Introduction and Objectives Asthma is a heterogeneous disease affecting 150–300 million people. Underlying mechanisms remain unclear. T H 17 cells expressing interleukin-17 are widely hypothesised to play a role, particularly in severe neutrophilic asthma. Mucosal associated invariant T (MAIT) cells are novel innate-like T-cells of unknown function which express CD161 and an invariant TCRα chain (Vα7.2-Jα33) and recognise the highly conserved restriction molecule MR1. We undertook to analyse IL-17 and key T-cell subsets, namely T H 17, T H 1, T H 2, T REG, and MAIT cells in relation to asthma severity and virus-induced exacerbations. Methods Cross sectional study: 76 subjects underwent detailed phenotyping, sputum induction, phlebotomy, and bronchoscopy. Samples were analysed by 9-colour flow-cytometry, RT-PCR, multiplex ELISA, microarray, and deep sequencing of the airway microbiome. Longitudinal study: 35 frequently exacerbating asthmatics: followed at 7 time-points during a naturally occurring cold. Results Contrary to initial hypotheses TH17 cell frequencies did not differ between health and any asthmatic phenotype, in any tissue compartment. TH2 cell frequencies were elevated in asthma in bronchoalveolar-lavage (BAL) (ANOVA p=0.041) and markedly in bronchial biopsies (p=0.048), as expected[1]. BAL TH1 cell frequencies were also increased in asthma (p=0.01) as described[2], whilst TREG frequencies were lower in severe asthma (p=0.019). T H 2 cytokines were increased in asthma in sputum (IL-5 p=0.005) and BAL (IL-5 p Longitudinal follow-up revealed no significant differences in T-cell frequencies during exacerbations, though sputum T H 17 cells tended to increase (NS). We observed that frequencies of Vα7.2+CD161+ (MAIT) cells in blood are lower in asthma than in health (p=0.013), and correlated with severity in blood (p for linear trend Conclusions A role for TH17 cells in asthma, particularly severe neutrophilic disease has been widely hypothesised, but is not supported by these data. High BAL IL-17 levels in older, steroid-naive, mild asthmatics may have a different cellular source. We describe a novel finding of deficient Vα7.2+CD161+ (MAIT) cells in severe asthma. References Robinson, DS, et al., Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med, 1992. 326(5): p. 298–304. Krug, N, et al., T-cell cytokine profile evaluated at the single cell level in BAL and blood in allergic asthma. Am J Respir Cell Mol Biol, 1996. 14(4): p. 319–26.