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Cigarette smoking has multiple serious negative health consequences. However, the epidemiological relationship between cigarette smoking and SARS-CoV-2 infection is controversial; and the interaction between cigarette smoking, airway expression of the ACE2 receptor and the susceptibility of airway cells to infection is unclear. We exposed differentiated air-liquid interface cultures derived from primary human airway stem cells to cigarette smoke extract (CSE) and infected them with SARS-CoV-2. We found that CSE increased expression of full-length ACE2 (flACE2) but did not alter the expression of a Type I-interferon sensitive truncated ACE2 that lacks the capacity to bind SARS-CoV-2 or a panel of interferon-sensitive genes. Importantly, exposure to CSE did not increase viral infectivity despite the increase in flACE2. Our data are consistent with epidemiological data suggesting current smokers are not at excess risk of SARS-CoV-2 infection. This does not detract from public health messaging emphasising the excess risk of severe COVID-19 associated with smoking-related cardiopulmonary disease.

BackgroundThere are limited effective prophylactic treatments for SARS-CoV-2 infection, and limited early treatment options. Viral cell entry requires spike protein binding to the ACE2 receptor and spike cleavage by TMPRSS2, a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is already in clinical trials in early SARS-CoV-2 infection.MethodsThe likely initial cells of SARS-CoV-2 entry are the ciliated cells of the upper airway. We therefore used differentiated primary human airway epithelial cells maintained at the air-liquid interface (ALI) to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection.ResultsWe first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-Cov-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, camostat is rapidly metabolised in the circulation in vivo, and systemic bioavailability after oral dosing is low. We therefore modelled local airway administration by applying camostat to the apical surface of the differentiated ALI cultures. We demonstrated that a brief exposure to topical camostat is effective at restricting SARS-CoV-2 viral infection.ConclusionThese experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial.

Background Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. Objective We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology; namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents which normally induce eosinophil apoptosis. Methods Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry, and GCS efficacy by apoptosis assays and qPCR. Results Hypoxic incubation (3 kPa) caused: (i) stabilisation of HIF-2α and up-regulation of hypoxia regulated genes including BNIP3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1), (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, that was most evident in eosinophils derived from atopic and asthmatic donors, (iii) enhanced F-actin formation, (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism), and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia. Conclusion and Clinical Relevance These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis and importantly, antagonising the normal pro-apoptotic effect of GCS. Since eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour these cells in vivo. This article is protected by copyright. All rights reserved.

BACKGROUND. Type 1 diabetes (T1D) results from loss of immune regulation, leading to the development of autoimmunity to pancreatic β cells, involving autoreactive T effector cells (Teffs). Tregs, which prevent autoimmunity, require IL-2 for maintenance of immunosuppressive functions. Using a response-adaptive design, we aimed to determine the optimal regimen of aldesleukin (recombinant human IL-2) to physiologically enhance Tregs while limiting expansion of Teffs. METHODS. DILfrequency is a nonrandomized, open-label, response-adaptive study of participants, aged 18–70 years, with T1D. The initial learning phase allocated 12 participants to 6 different predefined regimens. Then, 3 cohorts of 8 participants were sequentially allocated dose frequencies, based on repeated interim analyses of all accumulated trial data. The coprimary endpoints were percentage change in Tregs and Teffs and CD25 (α subunit of the IL-2 receptor) expression by Tregs, from baseline to steady state. RESULTS. Thirty-eight participants were enrolled, with thirty-six completing treatment. The optimal regimen to maintain a steady-state increase in Tregs of 30% and CD25 expression of 25% without Teff expansion is 0.26 × 106 IU/m2 (95% CI –0.007 to 0.485) every 3 days. Tregs and CD25 were dose-frequency responsive, Teffs were not. The commonest adverse event was injection site reaction (464 of 694 events). CONCLUSIONS. Using a response-adaptive design, aldesleukin treatment can be optimized. Our methodology can generally be employed to immediately access proof of mechanism, thereby leading to more efficient and safe drug development. TRIAL REGISTRATION. International Standard Randomised Controlled Trial Number Register, ISRCTN40319192; ClinicalTrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT02265809","term_id":"NCT02265809"}}NCT02265809. FUNDING. Sir Jules Thorn Trust, the Swiss National Science Foundation, Wellcome, JDRF, and NIHR Cambridge Biomedical Research Centre.

SummaryBackgroundType 1 diabetes (T1D) results from loss of immune regulation leading to the development of autoimmunity to pancreatic beta-cells, involving autoreactive T effector cells (Teffs). Regulatory T cells (Tregs), that prevent autoimmunity, require Interleukin-2 (IL-2) for maintenance of immunosuppressive functions and, alterations in the IL-2 pathway predispose to T1D. Using an adaptive trial design we aimed to determine the optimal regimen of aldesleukin (recombinant human IL-2) to physiologically enhance Tregs while limiting expansion of autoreactive Teffs.MethodsDILfrequency is a single-center, non-randomised, open-label, response-adaptive study of participants aged 18 to 70 years with T1D. The initial learning phase allocated 12 participants to six different predefined dose-frequency regimens. Then, three cohorts of 8 participants were sequentially allocated dose-frequencies, based on repeated interim analyses of all accumulated trial data. The co-primary endpoints were percentage change in Tregs, Teffs and, CD25 (α subunit of the IL-2 receptor) expression by Tregs, from baseline to steady state. Trial registration ISRCTN40319192 and ClinicalTrials.gov (NCT02265809).Findings115 participants were assessed between November 17th 2014 and May 22nd 2016, 38 participants were enrolled with 36 completing treatment. The optimal regimen to maintain a steady state increase in Tregs of 30% and CD25 expression of 25% without Teff expansion is 0.26 × 106 IU/m2 (95% CI (−0.007 to 0.485)) every 3 days (1.3 to 4.4). Tregs and CD25 were dose-frequency responsive, while Teffs were not. The commonest adverse event was injection site reaction (464/694 events), with a single participant developing transient eosinophilia at the highest dose (0.47 × 106 IU/m2).InterpretationThis response-adaptive trial defined a well-tolerated aldesleukin regimen that specifically induces Treg expansion that can now be trialled to treat T1D.FundingSir Jules Thorn Trust, Wellcome, JDRF, SNSF, NIHR

Eosinophils are mediators of allergic inflammation and are implicated in the pathogenesis of numerous conditions including asthma, parasitic infections, neoplasms, hyper-eosinophilic syndromes, vasculitic disorders, and organ-specific conditions. Assessing eosinophilic inflammation is therefore important in establishing a diagnosis, in monitoring and assessing response to treatment, and in testing novel therapeutics. Clinical markers of atopy and eosinophilic inflammation include indirect tests such as lung function, exhaled breath condensate analysis, fractional exhaled nitric oxide, serum immunoglobulin E levels and serum periostin. Direct measures, which quantify but do not anatomically localise inflammation include blood eosinophil counts, serum or plasma eosinophil cationic protein and sputum eosinophil levels. Cytology from bronchoalveolar lavage and histology from endobronchial and transbronchial biopsies are better at localising inflammation but are more invasive. Novel approaches using radiolabelled eosinophils with single-photon emission computed tomography, offer the prospect of non-invasive methods to localise eosinophilic inflammation.

The inflamed bronchial mucosal surface is a profoundly hypoxic environment. Neutrophilic airway inflammation and neutrophil-derived proteases have been linked to disease progression in conditions such as COPD and cystic fibrosis, but the effects of hypoxia on potentially harmful neutrophil functional responses such as degranulation are unknown.Following exposure to hypoxia (0.8% oxygen, 3 kPa for 4 h), neutrophils stimulated with inflammatory agonists (granulocyte-macrophage colony stimulating factor or platelet-activating factor and formylated peptide) displayed a markedly augmented (twofold to sixfold) release of azurophilic (neutrophil elastase, myeloperoxidase), specific (lactoferrin) and gelatinase (matrix metalloproteinase-9) granule contents. Neutrophil supernatants derived under hypoxic but not normoxic conditions induced extensive airway epithelial cell detachment and death, which was prevented by coincubation with the antiprotease α-1 antitrypsin; both normoxic and hypoxic supernatants impaired ciliary function. Surprisingly, the hypoxic upregulation of neutrophil degranulation was not dependent on hypoxia-inducible factor (HIF), nor was it fully reversed by inhibition of phospholipase C signalling. Hypoxia augmented the resting and cytokine-stimulated phosphorylation of AKT, and inhibition of phosphoinositide 3-kinase (PI3K)γ (but not other PI3K isoforms) prevented the hypoxic upregulation of neutrophil elastase release.Hypoxia augments neutrophil degranulation and confers enhanced potential for damage to respiratory airway epithelial cells in a HIF-independent but PI3Kγ-dependent fashion.

Radiolabelled leukocyte scans are widely used in nuclear medicine to locate sites of infection and inflammation. Radiolabelling of leukocyte subpopulations can also yield valuable information on cell trafficking and kinetics in vivo, but care must be taken to minimize inadvertent cell activation ex vivo. Here, we describe the use of autologous indium(111)-labelled eosinophils to measure eosinophil intravascular life-span and monitor their distribution and fate using gamma camera imaging in healthy non-atopic individuals.

Radiolabelled leukocyte scans are widely used in nuclear medicine to locate sites of infection and inflammation. Radiolabelling of leukocyte subpopulations can also yield valuable information on cell trafficking and kinetics in vivo, but care must be taken to minimize inadvertent cell activation ex vivo. Here, we describe the use of autologous indium(111)-labelled eosinophils to measure eosinophil intravascular life-span and monitor their distribution and fate using gamma camera imaging in healthy non-atopic individuals.

Patients with severe asthma make up a relatively small proportion of the total population of patients with asthma yet account for a disproportional amount of asthma-related morbidity and healthcare utilisation.1 2 These patients are usually highly symptomatic, difficult to treat and can be extremely refractory to current treatments. As a consequence, understanding the mechanisms underlying this particular form of asthma is of paramount importance. Morphological examination of the asthmatic airway reveals epithelial desquamation, thickening of the reticular basement membrane, mucus gland hyperplasia, goblet cell differentiation, angiogenesis and smooth muscle hypertrophy.3 In addition to these structural changes, an inflammatory cell infiltrate is evident within the airways comprising eosinophils, mast cells, lymphocytes and neutrophils. While eosinophils are the most characteristic inflammatory cell type present in mild to moderate asthma, the neutrophil seems to take centre stage more often in patients with severe disease. In fact, the neutrophil is one of the earliest inflammatory cells recruited to the airways following allergen exposure and is particularly evident in bronchoalveolar lavage samples and bronchial and transbronchial biopsies from patients with corticosteroid-resistant asthma, occupational asthma and fatal asthma as well as those with acute exacerbations.4–6 Furthermore, measures of asthma severity such as forced expiratory volume in 1 s correlate directly with the number of neutrophils present in the sputum and bronchial wall.7 8 There is also evidence of neutrophil activation and impaired function in the asthmatic airway.9 10 Moreover, neutrophil elastase is recognised to be highly histotoxic and results in epithelial and eosinophil activation, increased vascular permeability and promotion of transforming growth factor β release which is linked to airways remodelling. Given the propensity of neutrophils to die quite readily by ‘constitutive’ (ie, inbuilt time-dependent) apoptosis when studied in vitro,11 one major puzzle …

Introduction and objectives Neutrophils (PMNs) are a key component of the innate immune response to invading pathogens. They accumulate at sites of inflammation and infection, which are typically characterised by low oxygen tensions (e.g. in the acute respiratory distress syndrome (ARDS)). Human PMNs undergo constitutive apoptosis, their survival contingent upon pro-survival and pro-apoptotic signals derived from their microenvironment. Hypoxia profoundly delays PMN apoptosis, resulting in persistence of PMNs at inflammatory foci and this may perpetuate hypoxia-mediated lung injury. Given the importance of phosphoinositide 3-kinase (PI3-K) signalling in cytokine-mediated neutrophil survival, we hypothesised that hypoxia-induced PMN survival may also involve PI3-K-mediated signalling. Methods Highly pure PMNs isolated from healthy volunteers were incubated for 20 h under normoxic (20 kPa) and physiologically relevant hypoxic (3 kPa) conditions with a pan-PI3-K inhibitor (LY294002 at 10 μM), a novel pan-Class I PI3-K inhibitor (ZSTK474 at 1 μM, 3 μM and 10 μM) or novel PI3-K Class I isoform-selective inhibitors (PI3-Kδ at 1 μM; PI3-Kγ at 3 μM and 10 μM, or PI3-Kδγ at 3 μM). PMNs were also incubated in normoxia and hypoxia in the presence of GM-CSF (1 ng/ml) with the same panel of inhibitors, allowing comparison with GM-CSF mediated survival, which is largely PI3-K dependent. PMN apoptosis was assessed using two complementary techniques – morphology and flow cytometry following annexin V-FITC and propidium iodide staining. Results Compared with normoxia, hypoxia promoted PMN survival (mean% ± SEM apoptotic cells at 20 h; 30.9 ± 1.9 vs. 59.0 ± 1.8 respectively, p Conclusions Our results indicate that hypoxia-induced PMN survival is PI3-K dependent. Targeting this pathway may accelerate PMN apoptosis, resulting in resolution of inflammation.

Sites of infection and inflammation are profoundly hypoxic, requiring neutrophils to function under low oxygen tensions. Although neutrophils are well adapted and can rely on glycolytic metabolism, hypoxia still impairs the neutrophil oxidative burst, reduces bacterial killing and delays apoptosis. 1 As neutrophil proteases have been implicated in lung diseases such as COPD, we hypothesised that hypoxia might also promote neutrophil degranulation, with an enhanced potential for neutrophil-mediated tissue injury. Neutrophils isolated from healthy volunteers were subjected to normoxia (20 kPa) or hypoxia (3 kPa) and subsequently activated with GM-CSF (10 ng/ml) and the bacterial tri-peptide fMLP (100 nM). A549 cells and ciliated human primary bronchial epithelial (NHBE) cells were exposed to neutrophil supernatants, the extent of cellular damage was determined by MTT assay (A549 cells), EM ultrastructure and cleaved caspase 3 staining. Ciliary function was also investigated using video microscopy in the ciliated NHBE cells. Hypoxic incubation for 4 hours resulted in a 3–5 fold increase in neutrophil degranulation; this was evident for active elastase, MPO, MMP-9 and lactoferrin and hence occurred from all granule sub-types. Supernatants from hypoxic neutrophils induced 50% more cell death in A549 cells compared to supernatants from normoxic neutrophils. NHBE cells exposed to supernatants from hypoxic versus normoxic neutrophils suffered more cellular damage (EM; images were scored for cytoplasmic blebbing, vacuole formation and dead cells), an increase in LDH activity (from 35.7 ± 6 to 50.2 ± 0.7 nmol/min/ml, was indicative of cell death), increased cleaved caspase 3 staining was shown to be an indicator of apoptosis and there was a substantial increase in the proportion of dyskinetic and immotile cilia. In conclusion; hypoxia induced a destructive neutrophil phenotype with delayed apoptosis, impaired bacterial killing and increased release of histo-cytotoxic proteases. This phenotype may be important for understanding the mechanisms of chronic inflammatory diseases like COPD. Funded by the British Lung Foundation and NIHR Cambridge BRC. References McGovern, N. N. et al . Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils. J. Immunol. Baltim. Md 1950 186, 453–463 (2011).

Many tissues including the skin, intestinal epithelium and potentially the airway operate at ‘physiological’ levels of tissue hypoxia with normal P O 2 values below 3KPa. Both sterile and non-sterile inflammation exacerbates the degree of tissue hypoxia and predicates the need for granulocytes including eosinophils to operate efficiently under hypoxia. In these experiments we have examined the effects of hypoxia on eosinophil longevity and show that a P O 2 below 3KPa severely attenuates the pro-apoptotic effect of dexamethasone. Human blood eosinophils were prepared from healthy donors using hetastarch-sedimentation and EasySep ® -immunomagenetic beads, and cultured in RPMI + 10% autologous serum for 6–24 h. Apoptosis and efferocytosis were quantified using standard morphology, AnV/PI staining and myeloperoxidase counter-stain methods. Hypoxic incubation (H) (media P O 2 2.9±0.1 KPa) caused a marked survival response in eosinophils compared to normoxia (N) (% apoptosis at 24 h: N 16.3±3.0%; H 1.2±0.2%, n=5), which was of similar magnitude to that observed with IL-5 (N 4.2±0.3%). This hypoxic survival effect was mimicked by the iron chelator/2-oxyglutarate analogue DFO (10 mM) and DMOG (1 mM) and blocked in a concentration-dependent manner by the protein synthesis inhibitor cycloheximide. In preliminary experiments hypoxia also independently reduced the capacity of monocyte-derived macrophages to phagocytose apoptotic eosinophils as seen previously with neutrophils. Most strikingly, hypoxic incubation also reduced the normal pro-apoptotic effect of dexamethasone (1 µM) (% apoptosis at 24 h: N + Dex 27.6±3.9%, H + Dex 11.2±2.9%). qPCR analysis of the glucocorticosteroid-dependent gene GILZ and the hypoxia-HIF-1α-dependent gene GLUT1 demonstrated that the above suppression of dex-induced eosinophil apoptosis was not a consequence of the inhibition of steroid-induced transcriptional activity (mRNA fold change at 16 h: GILZ N 12.89±3.6, GILZ H 23.9±9.2; GLUT1 H (control) 28.5±7.2, GLUT1 H (Dex) 45.5±13.5, n=3). These data suggest that hypoxia can cause a profound effect on eosinophil longevity and macrophage uptake, and render these cells partially resistant to the pro-apoptotic effects of dexamethasone. This may impede the effective resolution of eosinophilic inflammation in vivo.

Tissues such as the skin and intestinal epithelium experience physiological hypoxia whereas pathological hypoxia occurs at inflammatory sites. Neutrophils are recruited to infective/inflamed areas and are thus required to operate under low oxygen tensions. We have shown previously that hypoxia delays neutrophil apoptosis (JEM 2005; 201:105) and impairs bacterial killing (J Immunol 2011; 186:453) and have now studied the effect of hypoxia on the release of histotoxic neutrophil proteases. Neutrophils isolated from healthy volunteers were subjected to normoxia or hypoxia (3 kPa). Superoxide anion release was measured by the reduction of cytochrome. C Elastase release was quantified by the cleavage of labelled elastin. Hypoxic incubation for 4 hours resulted in a 3- fold reduction in superoxide anion release from cells stimulated with GM-CSF and fMLP. In contrast, elastase release from the azurophilic granules was augmented almost 3-fold under hypoxia. The release of MMP-9 and lactoferrin was similarly up-regulated, suggesting a more generalised increase in degranulation under hypoxia. In addition to this electron microscopy showed that hypoxia induced a more activated phenotype (e.g. increased membrane ruffling and cell spreading). We show that hypoxia can induce a more destructive neutrophil phenotype, with enhanced degranulation and release of potentially histotoxic proteases, impaired bacterial killing, and delayed apoptosis. These data suggest that hypoxia aversively affects neutrophil function and may augment neutrophil mediated tissue destruction.