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Background: effective treatment for severe asthma is a significant unmet need. While eosinophilic inflammation caused by Type 2 cytokines is responsive to corticosteroid and biologic therapies, many severe asthmatics exhibit corticosteroid‐unresponsive mixed granulocytic inflammation. Objective: here, we tested the hypothesis that the pro‐allergic cytokine, IL‐13, can drive both corticosteroid‐sensitive and corticosteroid‐resistant responses. Results: by integration of in vivo and in vitro models of IL‐13 driven inflammation, we identify a role for the epidermal growth factor receptor (EGFR/ERBB1) as a mediator of corticosteroid‐unresponsive inflammation and bronchial hyperresponsiveness (BHR) driven by IL‐13. Topological data analysis using human epithelial transcriptomic data from the U‐BIOPRED cohort identified severe asthma groups with features consistent with the presence of IL‐13 and EGFR/ERBB activation, with involvement of distinct EGFR ligands. Our data suggest that IL‐13 may play a dual role in severe asthma: on the one hand driving pathologic corticosteroid‐refractory mixed granulocytic inflammation, but on the other hand underpinning beneficial epithelial repair responses, which may confound responses in clinical trials. Conclusion and clinical relevance: detailed dissection of those molecular pathways that are downstream of IL‐13 and utilize the ERBB receptor and ligand family to drive corticosteroid refractory inflammation should enhance the development of new treatments that target this sub‐phenotype(s) of severe asthma, where there is an unmet need.

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic interstitial lung disease. Median survival is only 3 years, and treatment options are limited. IPF is thought to be a result of a combination of genetic and environmental factors with repetitive micro-injuries to alveolar epithelial cells playing a central role. IPF is characterised by aberrant extra cellular matrix (ECM) deposition by activated myofibroblasts. Epithelial-mesenchymal transition (EMT) is a process where polarised epithelial cells undergo molecular changes allowing them to gain a mesenchymal phenotype, with a subsequent enhanced ability to produce ECM components and increased migration and/or invasion. The source of myofibroblasts in IPF has been debated for many years, and EMT has been proposed as a source of these cells. However, lineage tracing in transgenic mice suggests the contribution of epithelial cells, which have undergone EMT, to the fibroblast population may be negligible. Instead, recent findings suggest that alveolar epithelial type II (ATII) cells undergoing EMT promote a pro-fibrotic microenvironment through paracrine signalling activating local fibroblasts. This review paper explores the contribution of ATII cells, which have undergone EMT, in the context of pulmonary fibrosis.

Rationale: ADAMs (proteinases with a disintegrin and a metalloproteinase domain) have not been well studied in chronic obstructive pulmonary disease (COPD).Objectives: To investigate whether ADAM9 ...

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in airway epithelial cells for SARS-CoV-2. ACE2 binding to the SARS-CoV-2 protein spike triggers viral fusion with the cell plasma membrane, resulting in viral RNA genome delivery into the host. Despite ACE2’s critical role in SARS-CoV-2 infection, full understanding of ACE2 expression, including in response to viral infection, remains unclear. ACE2 was thought to encode five transcripts and one protein of 805 amino acids. In the present study, we identify a novel short isoform of ACE2 expressed in the airway epithelium, the main site of SARS-CoV-2 infection. Short ACE2 is substantially upregulated in response to interferon stimulation and rhinovirus infection, but not SARS-CoV-2 infection. This short isoform lacks SARS-CoV-2 spike high-affinity binding sites and, altogether, our data are consistent with a model where short ACE2 is unlikely to directly contribute to host susceptibility to SARS-CoV-2 infection.

Background: Altered collagen architecture with increased “bone-type” pyridinoline collagen cross-linking, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in Idiopathic Pulmonary Fibrosis (IPF). We recently identified that hypoxia inducible factor (HIF) pathway activation promotes induction of the bone-type collagen cross-linking enzymes lysyl hydroxylase 2 and lysyl oxidase-like 2, yet the consequences of this on collagen structure-function in lung fibrosis remain unknown. Methods: Using a long term 3D in vitro model of the fibroblastic focus we cultured primary lung fibroblasts from IPF donors without or with the HIF-stabilising compound IOX2. Hydroxyproline and mature pyridinium cross-links were measured by colorimetric assays, and collagen ultrastructure assessed by electron microscopy (EM). The biomechanical effects of HIF stabilisation were investigated by parallel plate compression testing. Results: IOX2 stabilised HIF within the 3D fibroblastic focus model, promoting HIF pathway activation to disproportionately induce collagen-modifying enzymes relative to collagen fibril synthesis. After 6 weeks of culture, mature pyridinium cross-links were significantly increased by IOX2 to levels we have previously observed in IPF tissue. Ultrastructural analysis of the collagen fibrils with EM identified that IOX2 significantly reduced fibril diameter to sizes comparable with collagen fibrils enzymatically extracted from IPF tissue. IOX2 induced a greater than 3-fold increase in tissue stiffness. Conclusions: HIF pathway activation is a core regulator of bone-type collagen fibrillogenesis and altered structure-function in lung fibrosis.

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in the airways for SARS-CoV-2. ACE2 binding to SARS-CoV-2 protein Spike triggers viral fusion with the cell membrane, resulting in viral RNA genome delivery into the host. Despite ACE2’s critical role in SARS-CoV-2 infection, an understanding of ACE2 expression, including in response to viral infection, remains unclear.Until nowACE2was thought to encode five transcripts and one 805 amino acid protein. Here we identify a novel short isoform of ACE2. ShortACE2is expressed in the airway epithelium, the main site of SARS-CoV-2 infection; it is substantially upregulated in response to interferon stimulation and RV infection, but not in response to SARS-CoV-2 infection, and it shows differential regulation in asthma patients. This short isoform lacks SARS-CoV-2 spike glycoprotein high-affinity binding sites and altogether, our data are consistent with a model where shortACE2may influence host susceptibility to SARS-CoV-2 infection.

Dendritic cells (DCs) play a central role in regulating innate and adaptive immune responses. It is well accepted that their regulatory functions change over the life course. In order to study DCs function during early life it is important to characterize the function of neonatal DCs. However, the availability of neonatal DCs is limited due to ethical reasons or relative small samples of cord blood making it difficult to perform large-scale experiments. Our aim was to establish a robust protocol for the generation of neonatal DCs from cord blood derived CD34+ hematopoietic stem cells. For the expansion of DC precursor cells we used a cytokine cocktail containing Flt-3 L, SCF, TPO, IL-3 and IL-6. The presence of IL-3 and IL-6 in the first 2 weeks of expansion culture was essential for the proliferation of DC precursor cells expressing CD14. After 4 weeks in culture, CD14+ precursor cells were selected and functional DCs were generated in the presence of GM-CSF and IL-4. Neonatal DCs were then stimulated with Poly(I:C) and LPS to mimic viral or bacterial infections, respectively. Poly(I:C) induced a higher expression of the maturation markers CD80, CD86 and CD40 compared to LPS. In line with literature data using cord blood DCs, our Poly(I:C) matured neonatal DCs cells showed a higher release of IL-12p70 compared to LPS matured neonatal DCs. Additionally, we demonstrated a higher release of IFN-γ, TNF-α, IL-1β and IL-6, but lower release of IL-10 in Poly(I:C) matured compared to LPS matured neonatal DCs derived from cord blood CD34+ hematopoietic stem cells. In summary, we established a robust protocol for the generation of large numbers of functional neonatal DCs. In line with previous studies, we showed that neonatal DCs generated form CD34+ cord blood progenitors have a higher inflammatory potential when exposed to viral than bacterial related stimuli., Highlights • A robust protocol for the generation of high numbers of neonatal dendritic cells. • IL-3 and IL-6 are crucial for the proliferation of cord blood CD34+ progenitors. • Neonatal DCs have a higher inflammatory potential when exposed to viral stimuli. • LPS induces higher release of IL-10 in neonatal DCs compared to Poly(I:C).

Background/Aim: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. Materials and Methods: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). Results: The outgrowths formed as a multicellular sheet with motile cilia becoming evident as the Matrigel was remodeled to provide an air interface; cultures were viable for more than one year. Immunofluorescence and electron microscopy (EM) identified an upper layer of mucociliary epithelium and a lower layer of highly organized extracellular matrix (ECM) interspersed with fibroblastic cells separated by a basement membrane. EM analysis of the mucosal construct after repetitive exposure of to CSE revealed epithelial damage, loss of cilia, and ECM remodeling, as occurs in vivo.Conclusions: We have developed a robust bronchial mucosal model. The structural changes observed following CSE exposure suggest the model should have utility for drug discovery and preclinical testing, especially those targeting airway remodeling.

Background: Altered collagen architecture, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in idiopathic pulmonary fibrosis (IPF), with the collagen cross-linking enzymes lysyl hydroxylase 2 (LH2) and lysyl oxidase-like 2 (LOXL2) promoting pathological bone-type collagen crosslinking and increased tissue stiffness (Jones et al eLife. 2018;7:e36354). However, the upstream mechanisms regulating this process remain unknown. Objective: To determine the cellular provenance of LOXL2 and LH2 in IPF tissue and identify the mechanisms promoting their dysregulation. Methods: Cellular provenance of LH2 and LOXL2 was analysed using mRNA in-situ hybridisation. IPF lung fibroblasts were cultured under a range of conditions that have been associated with IPF pathogenesis and their effects on LH2 and LOXL2 induction assessed by RTqPCR and Western Blotting. Small-interfering RNAs (siRNA) were transfected into IPF fibroblasts to investigate signalling pathways of interest and their role in regulating LH2 and LOXL2 expression. Results: LH2 and LOXL2 were co-expressed within fibroblastic foci in IPF tissue. Hypoxia-inducible factor (HIF) pathways were the strongest inducers of both LOXL2 and LH2 in IPF fibroblasts. siRNA mediated knockdown of HIF-1α but not HIF-2α prevented LH2 induction, whilst knockdown of both HIF-1α and HIF-2α was required to prevent LOXL2 induction. Conclusions: Our results suggest that HIF pathway activation via LH2/LOXL2 may be a core regulator of bone-type collagen fibrillogenesis in IPF.

Human rhinoviruses express 2 cysteine proteases, 2A and 3C, that are responsible for viral polyprotein processing. Both proteases also suppress host gene expression by inhibiting mRNA transcription, nuclear export and cap-dependent translation. However, the relative contribution that each makes in achieving this goal remains unclear. In this study we have compared both the combined and individual ability of the 2 proteases to shut downin cellulogene expression using a novel dynamic reporter system. Our findings show that 2A inhibits host gene expression much more rapidly than 3C. By comparing the activities of a representative set of proteases from the three different Human Rhinovirus (HRV) species, we also find variation in the speed at which host gene expression is suppressed. Our work highlights the key role that 2A plays in early suppression of the infected host cell response and shows that this can be influenced by natural variation in the activity of this enzyme.

Human rhinoviruses express 2 cysteine proteases, 2A and 3C, that are responsible for viral polyprotein processing. Both proteases also suppress host gene expression by inhibiting mRNA transcription, nuclear export and cap-dependent translation. However, the relative contribution that each makes in achieving this goal remains unclear. In this study we have compared both the combined and individual ability of the 2 proteases to shut down in cellulo gene expression using a novel dynamic reporter system. Our findings show that 2A inhibits host gene expression much more rapidly than 3C. By comparing the activities of a representative set of proteases from the three different Human Rhinovirus (HRV) species, we also find variation in the speed at which host gene expression is suppressed. Our work highlights the key role that 2A plays in early suppression of the infected host cell response and shows that this can be influenced by natural variation in the activity of this enzyme.

Background Maternal allergy is a strong risk factor for developing asthma and airway hyperresponsiveness (AHR). ADAM33 has been identified as an asthma susceptibility gene and is associated with AHR and impaired lung function in early life. Our aim was to investigate the effects of maternal murine allergic airway inflammation on the lungs of offspring before and after birth. We hypothesised that the effects of maternal allergy will be modified in Adam33 knock out (KO) compared to wild-type (WT) offspring. Methods Allergic airway inflammation in pregnant heterozygous (Adam33±) mice was induced by exposure to house dust mite (HDM) through intranasal challenges during pregnancy. Control mice were challenged with saline. WT (Adam33+/+) and KO (Adam33-/-) offspring from the same litters were studied on embryonic day (ED)17.5 and 2 or 4 weeks post partum (pp). Lung function was measured in response to increasing doses of methacholine and bronchoalveolar lavage fluid (BALF) was collected for differential cell counts. Lung tissue was obtained for RTqPCR, Western blot and immunohistochemistry. Results At 4 weeks pp, WT offspring of HDM challenged mothers showed significantly enhanced AHR compared to WT offspring of control mothers. KO of Adam33 protected against AHR in the offspring of allergic mothers. Adam33 mRNA expression was significantly enhanced in WT lungs of HDM challenged mothers at ED17.5, but unchanged pp. Differential cell counts in the BALF and mRNA expression of inflammatory mediators indicated an absence of allergic airway inflammation in all of the offspring. Remodelling genes were not affected at any time point studied. In contrast, Cholinergic Receptor Muscarinic 1 (Chrm1) mRNA was increased at 4 weeks in all offspring of HDM challenged mothers. Conclusions This study identifies an in utero gene-environment interaction involving Adam33. This interaction has implications for the subsequent development of AHR in early life. Further studies are needed to elucidate the precise mechanism(s) whereby ADAM33 mediates its effects. Our data suggest modulation of the contractility of the airways, possibly involving muscarinic receptors.

Introduction Most asthma has its origin in early life and probably involves gene-environment interactions. The asthma susceptibility gene ADAM33 is associated with bronchial hyperresponsiveness (BHR) and reduced lung function in young children. It encodes a membrane-anchored metalloprotease, which is shed as a soluble protein (sADAM33) whose levels are increased in asthma. We have previously shown that sADAM33, promotes airway remodelling and augments allergic airway inflammation in juvenile mice (Davies ER et al, JCI Insight 2016). This might be initiated by ADAM33 induced innate lymphocytes (Kelly JFC et al, Thorax 2017). The aim of this work was to evaluate the effect of sADAM33 on the allergic airway responses of neonates. Methods Human sADAM33 was induced in lungs of double transgenic (Ccsp/ADAM33) (dTg) mice from in utero up to 4 weeks post-partum. dTg mice or single transgenic (sTg) controls were challenged with house dust mite extract (HDM) 3 times a week for 2 weeks from 3 or 14 days post-partum. BHR and inflammation were quantified. Lung tissue was analysed by RT-qPCR and immunohistochemistry (IHC). Results After HDM challenge from day 3, Type 2-responsive genes Il-5, Ccl11/Eotaxin and Muc5ac were significantly increased. Whilst an increase in BHR was observed after HDM challenge, there was no significant difference between sADAM33-expressing and control mice. In contrast, when challenged from day 14, sADAM33-expressing mice had a more robust eosinophilic inflammatory response in the bronchoalveolar lavage fluid with increased Il-5 and Ccl11/Eotaxin mRNA expression compared to littermate controls. This was also associated with increased Acta2 mRNA expression and BHR. Conclusion These data indicate that sADAM33 does not augment allergic responses in neonatal mice as robustly as in older mice. This suggests that the immune microenvironment in neonates is not sufficiently mature to respond to the pro-allergic effects of sADAM33 that may induce innate lymphocytes to make the airways more susceptible to allergic airway inflammation.

Rationale ADAM33 is an asthma susceptibility gene that plays a role in both airway remodelling and susceptibility to allergic airways disease. To study the role of ADAM33 in asthma we have exposed an Adam33 Knockout (KO) mouse to a house dust mite (HDM) sensitisation and challenge protocol. We have found that these mice exhibit less remodelling, bronchial hyperresponsiveness (BHR) and eosinophilic inflammation than wild type (WT) mice (ER Davies et al., JCI-Insight 2016), however the mechanisms which contribute to this protective phenotype are not well understood. Methods To study how the response to HDM is altered by loss of Adam33 we challenged WT and KO mice with HDM or saline and took whole lung RNA samples for next generation RNA sequencing (RNAseq). Gene set enrichment analysis was used to identify pathways and gene ontology terms associated with the differential response to HDM between KO and WT mice. Results Control WT and KO mice were found to have very similar gene expression profiles at baseline (5 differentially expressed genes, including Adam33, FDR p Discussion The alteration in the pulmonary metabolic gene signature may underpin a shift in immune cell activation and/or modification of smooth muscle energy expenditure during airway contraction. These changes may explain why the KO mouse is protected from both allergic responses and BHR. Further work will aim to identify the source of the different metabolic behaviour at a cellular level and to assess oxidative stress in lungs of normal and Adam33 KO mice.

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A disintegrin and metalloproteinase 33 (ADAM33) is an asthma susceptibility gene. A soluble, catalytically active form (sADAM33) has been identified in bronchoalveolar lavage fluid, levels of which correlate with disease severity. To model the role of sADAM33 in asthma, a transgenic mouse, expressing human sADAM33 was generated. sADAM33 mice were found to be more susceptible than controls to allergic airways disease. This led to the hypothesis that induction of sADAM33 expression changes the underlying immune microenvironment in the airway, promoting susceptiblity to allergic airways disease. To study this, RNA samples from whole lungs of sADAM33 mice and control mice were sequenced for differential expression analysis. Gene ontology identified predominantly immune-related biological processes associated with sADAM33 expression. Validation across a wider cohort of samples using RTqPCR confirmed the up-regulation of lymphocyte effector cell markers (Nkp46, Gzmb), as well as negative regulators of effector responses (Ido1, Pd-l1). Protein levels of GZMB measured by ELISA were significantly increased in sADAM33 mice. Analysis of lymphocyte populations by flow cytometry identified an increased population of CD3-, NKp46+, KLRG1+ positive lymphocytes, which may represent an altered phenotype of innate lymphocyte populations in the airway. The biology of innate lymphocytes is complex, but there is increasing support for their involvement in allergic disease. The altered phenotype of this cell population within the sADAM33 mice may play an important role in their increased susceptibility to allergic airways disease and warrants further investigation.

Maternal allergy is a strong risk factor for developing asthma and airway hyperresponsiveness (AHR). The asthma susceptibility gene ADAM33 is associated with AHR and impaired lung function in early life. Our aim was to investigate gene-environment interactions involving Adam33 by determining the effects of maternal murine allergic airway inflammation on the lungs of offspring. We hypothesised that this will be modified in Adam33 knock out (KO) compared to wild-type (WT) offspring. Allergic airway inflammation was induced by exposure to house dust mite (HDM) allergen in pregnant heterozygous Adam33 mice. WT and KO offspring from the same litters were studied on embryonic day (ED)17.5 and 2 and 4 weeks post partum. Lung function was performed in response to methacholine, bronchoalveolar lavage fluid (BALF) collected for differential cell counts and lung tissue for RTqPCR. Compared with saline challenged mothers, 4-week-old WT offspring of HDM challenged mothers showed significantly enhanced AHR; KO offspring of HDM challenged mothers were protected against development of AHR. No inflammatory genes were up-regulated in the lungs and no inflammatory cells were present in the BALF at 2 or 4 weeks. While typical remodelling genes were not up-regulated at the time points studied, Adam33 mRNA expression was significantly increased in WT lungs at ED17.5 and Cholinergic Receptor Muscarinic 1 (Chrm1) mRNA at 4 weeks in offspring of HDM challenged mothers. This study identifies an in utero gene-environment interaction involving ADAM33 that has implications for the development of AHR in early life. Further studies are needed to elucidate the mechanisms whereby ADAM33 mediates this.

Rational A disintegrin and metalloproteinase 33 (ADAM33) was discovered in 2002 as an asthma susceptibility gene. Genetic associations have been made between ADAM33 polymorphisms and asthma disease severity, bronchial hyperresponsivenss (BHR) and rate of lung function decline in both adults and children. A soluble, catalytically active form of the protein (sADAM33) has been identified in the bronchoalveolar lavage fluid of patients, levels of which correlate with disease severity (Lee JY et al, AJRCCM 2006). To study the role sADAM33, a lung specific, doxycycline (Dox) inducible, transgenic mouse, expressing the human pro and metalloproteinase domains of the full-length protein was generated (Ccsp-rtTA/Otet-ADAM33-Pro-MP). Induction of sADAM33, followed by house dust mite (HDM) sensitisation and challenge, resulted in increased BHR and airway inflammation (Davies ER et al, JCI-Insight 2016). The mechanisms by which ADAM33 promotes this susceptibility are unclear. The aim of this work is to identify pathways that are augmented by the induction of sADAM33, which promote increased sensitivity to allergen. Methods RNA samples from whole lung of adult mice, where sADAM33 had been induced for 4 or 8 weeks, were analysed by next generation RNA sequencing. Identified genes were confirmed across experimental time points (72 hour, 7 day, 4 and 8 weeks on Dox) in wider sample cohorts of Ccsp-rtTA/Otet-ADAM33-Pro-MP and control mice through RTqPCR. Results The predominant signal from the RNAseq output was for modulation of immune response genes at 4 weeks of sADAM33 expression (GO:0006955 Immune response: 31 genes, 58.49% coverage, FDR p value=7.09 E-22). Genes associated with an immune activation signature (Ccl5, Irgm1, Gm12250, Gzmb, Ncr1) were validated at least one time point. By 8 weeks of sADAM33 expression genes associated with negative regulation of the response were also validated (Ido1, Cd274). Conclusion Induction of sADAM33 in murine lungs, without allergic sensitisation, augmented underlying immune processes in this transgenic mouse model, which may contribute to increased susceptibility to allergic airway inflammation and BHR when challenged with allergen. Further work is required to delineate how sADAM33 affects immune cell populations and their behaviour in the lung.

Inhibition of Pim1 kinase activity reduces viral replication by inducing the onset of bronchial epithelial cell death http://ow.ly/IJWGD

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.

Asthma is a common chronic disease characterized by variable respiratory distress with underlying airway inflammation and airflow obstruction. The incidence of asthma has risen inexorably over the past 50 years, suggesting that environmental factors are important in its etiology. All inhaled environmental stimuli interact with the lung at the respiratory epithelium, and it is a testament to the effectiveness of the airway innate defenses that the majority of inhaled substances are cleared without the need to elicit an inflammatory response. However, once this barrier is breached, effective communication with immune and inflammatory cells is required to protect the internal milieu of the lung. In asthma, the respiratory epithelium is known to be structurally and functionally abnormal. Structurally, the epithelium shows evidence of damage and has more mucus-producing cells than normal airways. Functionally, the airway epithelial barrier can be more permeable and more sensitive to oxidants and show a deficient innate immune response to respiratory virus infection compared with that in normal individuals. The potential of a susceptible epithelium and the underlying mesenchyme to create a microenvironment that enables deviation of immune and inflammatory responses to external stimuli may be crucial in the development and progression of asthma. In this review, we consider three important groups of environmental stimuli on the epithelium in asthma: oxidants, such as environmental pollution and acetaminophen; viruses, including rhinovirus; and agents that cause barrier disruption, such as house dust mite allergens. The pathology associated with each stimulus is considered, and potential future treatments arising from research on their effects are presented.

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.

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.

The bronchial epithelium and underlying fibroblasts form an epithelial mesenchymal trophic unit (EMTU) which controls the airway microenvironment. We hypothesised that cell-cell communication within the EMTU propagates and amplifies the innate immune response to respiratory viral infections.EMTU co-culture models incorporating polarized (16HBE14o-) or differentiated primary human bronchial epithelial cells (HBECs) and fibroblasts were challenged with double-stranded RNA (dsRNA) or rhinovirus. In the polarized EMTU model, dsRNA affected ionic but not macromolecular permeability or cell viability. Compared with epithelial monocultures, dsRNA-stimulated pro-inflammatory mediator release was synergistically enhanced in the basolateral compartment of the EMTU model, with the exception of IL-1alpha which was unaffected by the presence of fibroblasts. Blockade of IL-1 signalling with IL-1 receptor antagonist (IL-1Ra) completely abrogated dsRNA-induced basolateral release of mediators except CXCL10. Fibroblasts were the main responders to epithelial-derived IL-1 since exogenous IL-1alpha induced pro-inflammatory mediator release from fibroblast but not epithelial monocultures. Our findings were confirmed in a differentiated EMTU model where rhinovirus infection of primary HBECs and fibroblasts resulted in synergistic induction of basolateral IL-6 that was significantly abrogated by IL-1Ra. This study provides the first direct evidence of integrated IL-1 signalling within the EMTU to propagate inflammatory responses to viral infection.

AimsTo quantify the extent of squamous metaplasia in bronchial biopsies and relate it to the presence of chronic obstructive pulmonary disease (COPD), a smoking-related pathology.MethodsBronchial biopsies (n = 15 in each group) from smokers with COPD GOLD stage1 and GOLD stage2, smokers without COPD and healthy non-smokers were stained immunohistochemically with a panel of antibodies that facilitated the identification of pseudostratified epithelium and distinction of squamous metaplasia and squamous epithelium from tangentially cut epithelium. The percentage length of each of these epithelial phenotypes was measured as a percent of total epithelial length using computerised image analysis. Sections were also stained for carcinoembryonic antigen and p53, early markers of carcinogenesis, and Ki67, and the percentage epithelial expression measured.ResultsThe extent of squamous metaplasia was significantly increased in both COPD1 and COPD2 compared to healthy smokers and healthy non-smokers. The amount of fully differentiated squamous epithelium was also increased in COPD1 and COPD2 compared to healthy non-smokers, as was the expression of carcinoembryonic antigen. These features correlated with one other.ConclusionIn subjects with COPD there is a loss of pseudostratified epithelium accompanied by an increase in squamous metaplasia with transition into a fully squamous epithelium and expression of early markers of carcinogenesis.

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 'alveolar' cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham's F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

The asthma susceptibility gene, a disintegrin and metalloprotease-33 (ADAM33), is selectively expressed in mesenchymal cells, and the activity of soluble ADAM33 has been linked to angiogenesis and airway remodeling. Transforming growth factor (TGF)-β is a profibrogenic growth factor, the expression of which is increased in asthma, and recent studies show that it enhances shedding of soluble ADAM33. In this study, we hypothesized that TGF-β also affects ADAM33 expression in bronchial fibroblasts in asthma. Primary fibroblasts were grown from bronchial biopsies from donors with and those without asthma, and treated with TGF-β(2) to induce myofibroblast differentiation. ADAM33 expression was assessed using quantitative RT-PCR and Western blotting. To examine the mechanisms whereby TGF-β(2) affected ADAM33 expression, quantitative methylation-sensitive PCR, chromatin immunoprecipitation, and nuclear accessibility assays were conducted on the ADAM33 promoter. We found that TGF-β(2) caused a time- and concentration-dependent reduction in ADAM33 mRNA expression in normal and asthmatic fibroblasts, affecting levels of splice variants similarly. TGF-β(2) also induced ADAM33 protein turnover and appearance of a cell-associated C-terminal fragment. TGF-β(2) down-regulated ADAM33 mRNA expression by causing chromatin condensation around the ADAM33 promoter with deacetylation of histone H3, demethylation of H3 on lysine-4, and hypermethylation of H3 on lysine-9. However, the methylation status of the ADAM33 promoter did not change. Together, these data suggest that TGF-β(2) suppresses expression of ADAM33 mRNA in normal or asthmatic fibroblasts. This occurs by altering chromatin structure, rather than by gene silencing through DNA methylation as in epithelial cells. This may provide a mechanism for fine regulation of levels of ADAM33 expression in fibroblasts, and may self-limit TGF-β(2)-induced ectodomain shedding of ADAM33.

Background and objective: a disintegrin and metalloproteinase (ADAM) 33 is a susceptibility gene associated with inflammatory lung and skin diseases. It is selectively expressed in mesenchymal cells, and its metalloprotease activity has been linked to angiogenesis and tissue remodelling. A soluble form of ADAM33 (sADAM33) has been identified in the bronchoalveolar lavage fluid (BALF) of asthmatic patients, and its levels inversely correlate with lung function. Because of its association with inflammatory lung diseases, it was hypothesized that sADAM33 is elevated in BALF of patients with pulmonary sarcoidosis. Methods: after removal of Ig using Protein A/G and enrichment using Concanavalin A beads, sADAM33 was identified in BALF by Western blotting. A fluorescence resonance energy transfer peptide cleavage assay was used to assess ADAM33-like activity in BALF. Results: sADAM33 protein in BALF was detected as a 25 kDa fragment, and levels were significantly increased in samples from sarcoid patients when compared to healthy controls (P Conclusions: release of sADAM33 is increased in sarcoid and may be associated with abnormal lung function. sADAM33 may be a biomarker of lung tissue inflammation and remodelling in sarcoid

Background: Rhinovirus (RV)-induced chronic obstructive pulmonary disease (COPD) exacerbations exhibit TH2-like inflammation. We hypothesized that RV-infected bronchial epithelial cells (BEC) overproduce TH2-switching hub cytokine, thymic stromal lymphopoietin (TSLP) in COPD. Methods: Primary BEC from healthy (HBEC) and from COPD donors (COPD-BEC) were grown in 12-well plates, infected with RV16 (0.5–5 MOI) or stimulated with agonists for either toll-like receptor (TLR) 3 (dsRNA, 0.1–10 µg/ml) or RIG-I-like helicases (dsRNA-LyoVec, 0.1–10 µg/ml). Cytokine mRNA and protein were determined (RTqPCR; ELISA). Results: dsRNA dose-dependently evoked cytokine gene overproduction of TSLP, CXCL8 and TNF-α in COPD-BEC compared to HBEC. This was confirmed using RV16 infection. IFN-β induction did not differ between COPD-BEC and HBEC. Endosomal TLR3 inhibition by chloroquine dose-dependently inhibited dsRNA-induced TSLP generation and reduced generation of CXCL8, TNF-α, and IFN-β. Stimulation of cytosolic viral sensors (RIG-I-like helicases) with dsRNA-LyoVec increased production of CXCL8, TNF-α, and IFN-β, but not TSLP. Conclusions: Endosomal TLR3-stimulation, by dsRNA or RV16, induces overproduction of TSLP in COPD-BEC. dsRNA- and RV-induced overproduction of TNF-α and CXCL8 involves endosomal TLR3 and cytosolic RIG-I-like helicases and so does the generation of IFN-β in COPD-BEC. RV16 and dsRNA-induced epithelial TSLP may contribute to pathogenic effects at exacerbations and development of COPD.

Toxic injury can induce squamous metaplasia of respiratory epithelium, which normally is pseudostratified. Terminally differentiated squamous epithelial cells have a flattened, elongated appearance. During differentiation, they have an intermediate phenotype that is difficult to identify and distinguish from tangentially cut columnar cells in tissue sections from endobronchial biopsies, whose small size makes orientation difficult. The aim of our study was to develop a panel of antibodies that could be employed to distinguish normal epithelium from metaplastic epithelium and would be suitable for use on endobronchial biopsies. Nasal polyp tissue and tonsil tissue, which have pseudostratified and squamous epithelia, respectively, were collected from surgical cases and embedded in glycol methacrylate resin. Cut sections were stained immunohistochemically with a panel of antibodies to cytokeratins (CK), whose expression varies with epithelial type and stage of differentiation, and involucrin, a marker of terminal squamous differentiation. Squamous epithelium stained positively for CK5/6, CK13 and involucrin. In the pseudostratified epithelium, basal cells exhibited weak staining for CK13 and strong staining for CK5/6, and columnar cells exhibited strong immunoreactivity for CK7, CK8 and CK18. Application of this panel to endobronchial biopsies from smokers enabled areas of squamous metaplasia to be distinguished from tangentially sectioned epithelium.

Aims: Mutations in the FERM domain containing 7 (FRMD7) genes are known to cause a significant number of cases of congenital idiopathic nystagmus (CIN). Only limited expression data exist suggesting low levels of expression in all tissues. In this study, we assess the expression profile of the murine homologue of FRMD7(Frmd7) in tissue from three murine organs during development. Methods: cDNA was extracted from heart, lung, and brain tissues of MF-1 mice at 12 developmental time points, embryonic days 11–19, postnatal days 1 and 8, and from adult mice. Relative expression of Frmd7mRNA was calculated using quantitative real-time PCR techniques with two normalising genes (Gapdhand Actb). Results: Expression of Frmd7was low in all tissues consistent with earlier reports. In heart and lung tissues, expression remained very low with an increase only in adult samples. In brain tissue, expression levels were higher at all time points with a significant increase at embryonic day 18, with no gender-specific influence on Frmd7expression. Conclusions: Frmd7is expressed at low levels in all tissues studied suggesting a role in many tissue types. However, higher overall expression and a sharp increase at ED18 in the murine brain suggest a different role in this tissue. Earlier studies have shown that genes expressed in the murine brain during development exhibit temporal functional clustering. The temporal pattern of Frmd7 expression found in this study mirrors that of genes involved in synapse formation/function, and genes related to axon growth/guidance. This suggests a role for Frmd7 in these processes and should direct further expression studies.

Ciliated Bronchial Epithelium (CBE) 1 is a novel gene, which is expressed in ciliated cells. As cilia are important during embryogenesis, the present authors characterised the murine homologue of CBE1 (Cbe1) and compared its temporal expression during murine and human lung development. Cbe1 cDNA was cloned and characterised using sequencing, standard PCR and Western blotting. Mouse and human embryonic/fetal lungs (HELs) were harvested for mRNA analysis and protein localisation in vivo and in vitro using RT-PCR and immunohistochemistry. The Cbe1 amino acid sequence was >75% identical with CBE1 and its alternative splicing and tissue distribution were highly conserved. Pulmonary expression of Cbe1 mRNA was increased at embryonic day (E)16, 1 day later than Foxj1, which is consistent with a role in ciliogenesis. In HELs, CBE1 mRNA was detectable at 8-9 weeks post-conception and increased in explant culture. CBE1 protein expression was weak at 10 weeks post-conception but strong at 12.3 weeks post-conception, in parallel with cilia formation. Additionally, Cbe1 mRNA was expressed at E11 (4-5 weeks post-conception in HELs) in the absence of Foxj1, implying a distinct role in early development. Chronological regulation of CBE1/Cbe1 expression during pulmonary differentiation suggests involvement in ciliogenesis, with an additional role during early lung development.

Background IL-13 is a key cytokine associated with the asthmatic phenotype. IL-13 signals via its cognate receptor, a complex of IL-13 receptor (IL-13R) α 1 chain with IL-4 receptor α; however, a second protein, IL-13Rα2, also binds IL-13. Recently a polymorphic variant of IL-13 (R110Q) has been shown to be associated with atopy. Objective To investigate the binding properties of this IL-13 variant to its cognate receptors. Methods We used surface plasmon resonance to measure the binding kinetics of R110Q to its receptors. Primary human fibroblasts were grown from endobronchial biopsies obtained from volunteers. Receptor levels were measured by fluorescence-activated cell sorting. Results There was no significant difference in the binding of R110Q with soluble human IL-13Rα1 compared with IL-13 (32 ± 5 nmol/L and 36 ± 7 nmol/L, respectively; P = .625). However, a small but significant difference was observed in the binding of R110Q to soluble human IL-13Rα2 compared with IL-13 (840 ± 87 pmol/L and 1.1 ± .05 nmol/L, respectively; P = .04). We observed that primary human lung fibroblasts expressed different levels of IL-13Rα2. Eotaxin release from fibroblasts expressing low IL-13Rα2 levels was significantly higher in response to R110Q compared with IL-13. This was not evident in cells that had high baseline IL-13Rα2 levels. Conclusion These results suggest that relatively small changes in functional properties of a ligand combined with variation in receptor levels in vivo can result in significant differences in responsiveness. Clinical implications Expression of R110Q and low IL-13Rα2 levels can result in important biological differences that may have clinical relevance in an atopic environment.

Background Cysteinyl leukotrienes (cys-LTs) are potent asthma-related mediators that function through their G protein–coupled receptors, cys-LT receptor type 1 (CysLT1R) and cys-LT receptor type 2 (CysLT2R). Objective Because many G protein–coupled receptors transactivate the epidermal growth factor receptor (EGFR) through metalloprotease-mediated ligand shedding, we investigated the effects of cys-LTs on signal transduction and proliferation of bronchial fibroblasts. Methods Human bronchial fibroblasts were grown from biopsy specimens of healthy subjects. Mitogenesis was assessed on the basis of tritiated methylthymidine incorporation. Results Leukotriene (LT) D 4 alone did not increase mitogenesis but dose-dependently increased thymidine incorporation and cell proliferation in the presence of epidermal growth factor (EGF). The enhancement was not prevented by CysLT1R antagonists (MK-571 and montelukast) or by a dual antagonist (BAY u9773), which is consistent with the lack of detectable mRNA for CysLT1R and CysLT2R in bronchial fibroblasts. LTD 4 did not cause EGFR transphosphorylation nor was the synergism blocked by the metalloprotease inhibitor GM6001. The EGFR-selective kinase inhibitor AG1478 suppressed the synergy between LTD 4 and EGF but had no effect on synergistic interactions of LTD 4 with other receptor tyrosine kinase growth factors. The effect of LTD 4 involved a pertussis toxin–sensitive and protein kinase C–mediated intracellular pathway, leading to sustained growth factor–dependent phosphorylation of extracellular signal–regulated kinase 1/2 and protein kinase B (PKB/Akt). Conclusion Cys-LTs do not transactivate EGFR but have a broader capability to synergize with receptor tyrosine kinase pathways. Clinical implications This study implies a critical role of cys-LTs in airway fibrosis in asthma and other chronic airway diseases, which might not be blocked by therapy with current LT receptor antagonists.

Introduction: Respiratory viruses infect the airway epithelial barrier and cause inflammation. This process involves cellular crosstalk between different cell types. By using a dynamic 3D co-culture model of airway epithelial cells (AECs) and endothelial cells we aimed to analyse the cellular crosstalk regulating airway inflammation during viral infections. Methods: Human AECs were cultured on the apical side of permeable filter supports while endothelial cells were cultured on the basolateral side facing the microfluidic flow. The 3D model was challenged apically with double stranded RNA (Poly(I:C)), a mimic of viral infections. Basal supernatants were collected in 2h intervals and mediator secretion determined by ELISA. Results: In contrast to static cultures, the microfluidic systemallowed analysis of airway barrier responses time-dependently at shorter intervals and with greater sensitivity. Poly(I:C) induced release of TNF-α by epithelial cells with a peak 4h after stimulation; this was followed temporally by release of fractalkine (CX3 CL1), a chemokine regulating immune cell migration, from the endothelial cells. Release of fractalkine could be prevented by neutralisation of TNF-α. Conclusion: Using adynamic 3D model offers the potential to investigate mechanisms of airway inflammation and cellular crosstalk under conditions closer to the in vivo situation. During viral infections, endothelial-derived CX3 CL1 release is triggered by epithelial derived TNF-α. This cellular crosstalk might play an important role in the regulation of immune cell infiltration.

The airway epithelium is an important barrier interface for the lungs and plays a key role in various lung inflammatory disorders. MS4A8B, a member of the MS4A (membrane-spanning, 4 domains, subfamily A) family, contains a putative immunoreceptor tyrosine-based inhibitory motif (ITIM) and we have previously shown that MS4A8B is expressed on the cilia of airway epithelial cells. Our study aimed to determine the expression and function of MS4A8B in the human airways and to clarify its role in lung inflammatory disorders. Real-time PCR was performed on healthy and COPD primary bronchial epithelial cells (PBECs) grown at an air-liquid interface (ALI) to examine differences in MS4A8B expression. MS4A8B, and an ITIM mutant form, were transfected into the BEAS-2B and HEK293 cell lines to examine its effects on cellular function and signaling. MS4A8B gene expression increased over time as PBECs underwent complete mucociliary differentiation. Compared to healthy donors, MS4A8B gene expression was reduced in ALI cultured PBECs obtained from COPD patients. Transfected BEAS-2B cells stably expressing MS4A8B showed a reduced proinflammatory cytokine production and enhanced proliferation that was reversed by tyrosine mutation of the proteins ITIM motif. In signaling studies, MS4A8B was shown to be tyrosine phosphorylated in transfected HEK293 cells following sodium pervanadate treatment with this effect being lost upon MS4A8B ITIM mutation. Our combined data suggests that MS4A8B is an important control mechanism in the airway epithelium and that its ITIM motif is critical for this effect. The loss of MS4A8B expression in airway disease such as COPD may thus enhance airway inflammation and worsen disease.

Bronchial epithelial cells (BECs) and fibroblasts (FB) form an epithelial mesenchymal trophic unit (EMTU) that drives responses to disruptions in homeostasis such as viral infection. Evidence suggests that viral infection of BECs releases mediators which activate FBs. We hypothesised that epithelial virus exposure activates the EMTU. The EMTU was modelled using polarised BECs (16HBE14o-) and FBs (MRC5) maintained on the apical and basolateral surface of a nanoporous membrane respectively. Cytokine responses to dsRNA (a viral mimetic) were determined by ELISA. A primary EMTU model was established using primary BECs differentiated at an air-liquid interface, co-cultured with FBs and challenged with rhinovirus (RV16). dsRNA activation of the EMTU model induced significant IL-6, IL-8, GM-CSF and IP-10 release which was synergistically enhanced basolaterally compared to BEC or FB monocultures. dsRNA dependent IL-1αrelease was also detected in the EMTU model and BECs.IL-1 receptor (IL-1R) antagonist abrogated dsRNA-dependent basolateral IL-6, IL-8 and GM-CSF responses.However, only FBs were responsive to stimulation with exogenous IL-α. In the primary EMTU model, RV16 induced IL-1α,IL-6, IL-8 and IP-10 release and following IL-1R antagonism there was a significant reduction in RV16-dependent IL-6 and IL-8. In the EMTU cross-talk between BECs and FBs led to synergistic enhancement in viral-induced responses. Viral-induced basolateral inflammatory responses were mediated by BEC-derived IL-1αacting on FBs.IL-1α may therefore have important consequences in promoting inflammation in viral exacerbations of chronic lung diseases such as asthma.

The bronchial epithelium plays a key role in providing a protective barrier against many environmental substances of anthropogenic or natural origin which enter the lungs during breathing. Appropriate responses to these agents are critical for regulation of tissue homeostasis, while inappropriate responses may contribute to disease pathogenesis. Here, we compared epithelial barrier responses to different pollen species, characterized the active pollen components and the signaling pathways leading to epithelial activation. Polarized bronchial cells were exposed to extracts of timothy grass (Phleum pratense), ragweed (Ambrosia artemisifolia), mugwort (Artemisia vulgaris), birch (Betula alba) and pine (Pinus sylvestris) pollens. All pollen species caused a decrease in ionic permeability as monitored trans-epithelial electrical resistance (TER) and induced polarized release of mediators analyzed by ELISA, with grass pollen showing the highest activity. Ultrafiltration showed that the responses were due to components

BackgroundEpidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.ObjectivesSince primary bronchial epithelial cells (PBECs) from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.MethodsPBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE); cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF).ResultsExposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH), but not ascorbic acid (AA), protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective.ConclusionOur results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.

Background IL-13 and IL-4 share many functional properties as a result of their use of a common receptor complex comprising IL-13 receptor α 1 (IL-13Rα1) and IL-4 receptor α (IL-4Rα). The nonsignaling receptor IL-13 receptor α 2 (IL-13Rα2) binds IL-13 with high affinity and specificity and is believed to be a decoy receptor for IL-13. Objective We sought to examine the inhibitory effects of soluble and membrane-bound IL-13Rα2 on IL-13– and IL-4–mediated effects. Methods Primary human fibroblasts were grown from endobronchial biopsy specimens obtained from volunteers. Upregulation of IL-13Rα2 mRNA was measured by means of RT-PCR, and the level of surface expression was measured by means of FACS. Results We found that a recombinant soluble form of IL-13Rα2 blocked the effects of IL-13, but not IL-4, in fibroblasts in vitro . However, we found that the transmembrane form of IL-13Rα2 could attenuate both IL-13 and IL-4 responses, even though the response to TNF-α was unaffected. Furthermore, we found that IL-13Rα2 became associated with IL-4Rα in the presence of IL-4. Addition of a blocking antibody to the extracellular domain of IL-13Rα2 restored responses of both IL-13 and IL-4. Conclusion The ability of IL-13Rα2 to regulate IL-4 was previously unrecognized in primary airway cells. These data reveal a novel role for IL-13Rα2 as a negative regulator of both IL-13 and IL-4 signaling in human bronchial fibroblasts. Clinical implications It appears that IL-13Rα2 might be a powerful suppressor of T H 2-mediated responses and thus represents a potential therapeutic target for the treatment of asthma.

Induced sputum is a readily accessible biological fluid whose composition may alter as a consequence of disease. To date, however, the proteins that routinely populate this biofluid are largely unknown, in part due to the technical difficulties in processing such mucin-rich samples. To provide a catalogue of sputum proteins, we have surveyed the proteome of human-induced sputum (sputome). A combination of 2-D gel analysis and GeLC-MS/MS allowed a total of 191 human proteins to be confidently assigned. In addition to the expected components, several hitherto unreported proteins were found to be present, including three members of the annexin family, kallikreins 1 and 11, and peroxiredoxins 1, 2 and 5. Other sets of proteins identified included four proteins previously annotated as hypothetical or conserved hypothetical. Taken together, these data represent the first extensive survey of the proteome of induced sputum and provide a platform for future identification of biomarkers of lung disease.

IL-4 is a key cytokine associated with allergy and asthma. Induction of cell signaling by IL-4 involves interaction with its cognate receptors, a complex of IL-4Rα with either the common γ-chain or the IL-13R chain α1 (IL-13Rα1). We found that IL-4 bound to the extracellular domain of IL-4Rα (soluble human (sh)IL-4Rα) with high affinity and specificity. In contrast with the sequential mechanism of binding and stabilization afforded by IL-4Rα to the binding of IL-13 to IL-13Rα1, neither common γ-chain nor IL-13Rα1 contributed significantly to the stabilization of the IL-4:IL-4Rα complex. Based on the different mechanisms of binding and stabilization of the IL-4R and IL-13R complexes, we compared the effects of shIL-4Rα and an IL-4 double mutein (R121D/Y124D, IL-4R antagonist) on IL-4- and IL-13-mediated responses. Whereas IL-4R antagonist blocked responses to both cytokines, shIL-4Rα only blocked IL-4. However, shIL-4Rα stabilized and augmented IL-13-mediated STAT6 activation and eotaxin production by primary human bronchial fibroblasts at suboptimal doses of IL-13. These data demonstrate that IL-4Rα plays a key role in the binding affinity of both IL-13R and IL-4R complexes. Under certain conditions, shIL-4Rα has the potential to stabilize binding IL-13 to its receptor to augment IL-13-mediated responses. Thus, complete understanding of the binding interactions between IL-4 and IL-13 and their cognate receptors may facilitate development of novel treatments for asthma that selectively target these cytokines without unpredicted or detrimental side effects.

Background: Transforming growth factor beta (TGFβ) upregulates a number of smooth muscle specific genes in (myo)fibroblasts. As asthma is characterised by an increase in airway smooth muscle, we postulated that TGFβ 2 favours differentiation of asthmatic (myo)fibroblasts towards a smooth muscle phenotype. Methods: Primary fibroblasts were grown from bronchial biopsy specimens from normal (n = 6) and asthmatic (n = 7) donors and treated with TGFβ 2 to induce myofibroblast differentiation. The most stable genes for normalisation were identified using RT-qPCR and the geNorm software applied to a panel of 12 “housekeeping” genes. Expression of α-smooth muscle actin (αSMA), heavy chain myosin (HCM), calponin 1 (CPN 1), desmin, and γ-actin were measured by RT-qPCR. Protein expression was assessed by immunocytochemistry and western blotting. Results: Phospholipase A2 and ubiquitin C were identified as the most stably expressed and practically useful genes for normalisation of gene expression during myofibroblast differentiation. TGFβ 2 induced mRNA expression for all five smooth muscle related transcripts; αSMA, HCM and CPN 1 protein were also increased but desmin protein was not detectable. Although there was no difference in basal expression, HCM, CPN 1 and desmin were induced to a significantly greater extent in asthmatic fibroblasts than in those from normal controls (p = 0.041 and 0.011, respectively). Conclusions: Although TGFβ 2 induced the transcription of several smooth muscle related genes, not all were translated into protein. Thus, while TGFβ 2 is unable to induce a bona fide smooth muscle cell phenotype, it may “prime” (myo)fibroblasts for further differentiation, especially if the cells are derived from asthmatic airways.

Although asthma is defined in terms of reversibility of airflow obstruction, as the disease becomes more severe and chronic, it adopts different characteristics, including a degree of fixed airflow obstruction and corticosteroid refractoriness. Underlying these phenotypes is evidence of airway wall remodeling, which should be distinguished from the increase in smooth muscle linked to airways hyperresponsiveness. Aberrant epithelial-mesenchymal communication leads to a chronic wound scenario, which is characterized by activation of the epithelial-mesenchymal trophic unit, epithelial damage, the laying down of new matrix, and greater involvement of neutrophils in the inflammatory response. In allergic asthmatic patients who remain symptomatic despite high-dose corticosteroid therapy, blockade of IgE with omalizumab confers appreciable clinical benefit. Chronic severe asthma is also accompanied by a marked increase in TNF-α production that might contribute to corticosteroid refractoriness. Based on this, TNF blockade with the soluble fusion protein entanercept produces improvement in asthma symptoms, lung function, and quality of life paralleled by a marked reduction in airways hyperresponsiveness. Identification of novel susceptibility genes, such as a disintegrin and metalloprotease 33 (ADAM33), will provide further targets against which to direct novel therapies for asthma, especially at the more severe end of the disease spectrum.

Asthma is a complex disorder in which major genetic and environmental factors interact to both initiate the disease and modify its progression. While asthma is recognised as a disorder of the conducting airways characterised by Th2-directed inflammation, it is being increasingly apparent that alteration of the structural cells of the airways (airway remodelling) is also fundamental to disease chronicity and severity. The gene ADAM33, encoding a novel member of a identified as an asthma susceptibility gene as the result of a positional cloning effort in a cohort of families recruited form the UK and USA. Subsequent genetic studies have now provided evidence that ADAM33 may be involved in determining lung function throughout life, associated with early life lung function as well as increased decline therapeutic intervention in asthma and future work will focus on the mechanisms by which it alters lung function and bronchial hyperresponsiveness.

Asthma is increasing in prevalence in the developing world, affecting approximately 10% of the world's population. It is characterised by chronic lung inflammation and airway remodelling associated with wheezing, shortness of breath, acute bronchial hyperresponsiveness to a variety of innocuous stimuli and a more rapid decline in lung function over time. Airway remodelling, involving proliferation and differentiation of mesenchymal cells, particularly myofibroblasts and smooth muscle cells, is generally refractory to corticosteroids and makes a major contribution to disease chronicity. Transforming growth factor-beta is a potent profibrogenic factor whose expression is increased in the asthmatic airways and is a prime candidate for the initiation and persistence of airway remodelling in asthma. This review highlights the role of transforming growth factor-beta in the asthmatic lung, incorporating biosynthesis, signalling pathways and functional outcome. In vivo, however, it is the balance between transforming growth factor-beta and other growth factors, such as epidermal growth factor, which will determine the extent of fibrosis in the airways. A fuller comprehension of the actions of transforming growth factor-beta, and its interaction with other signalling pathways, such as the epidermal growth factor receptor signalling cascade, may enable development of therapies that control airway remodelling where there is an unmet clinical need.

The bronchial epithelium is constantly exposed to a wide range of environmental materials present in inhaled air, including noxious gases and anthropogenic and natural particulates, such as gas and particles from car emissions, tobacco smoke, pollens, animal dander, and pathogens. As a fully differentiated, pseudostratified mucociliary epithelium, the bronchial epithelium protects the internal milieu of the lung from these agents by forming a physical barrier involving adhesive complexes and a chemical barrier involving secretion of mucus, which traps inhaled particles that can be cleared by the mucociliary escalator. It is a testament to the effectiveness of these two barriers that most environmental challenges are largely overcome without the need to develop an inflammatory response. However, as the initial cell of contact with the environment, the bronchial epithelium also plays a pivotal role in immune surveillance and appropriate activation of immune effector cells and antigen presenting cells in the presence of pathogens or other danger signals. Thus, the bronchial epithelium plays a central role in controlling tissue homeostasis and innate immunity. This review will discuss these barrier properties and how dysregulation of these homeostatic mechanisms can contribute to disease pathologies such as asthma.

Asthma has a high heritability of up to 75%, involving a few genes with moderate effects rather than many genes with small effects. Two methods most frequently used to search for disease-causing genes are the candidate gene approach and positional cloning (reverse genetics). The candidate gene approach selects genes that seem to be relevant to the pathophysiology of a particular disease. Polymorphism in the genes is then directly tested for the genes’ involvement in the disease by association analyses. A second method is positional cloning, incorporating genome-wide scans. Here the entire genome is screened in a family-based cohort using a panel of DNA markers spaced at regular intervals across the genome to identify specific chromosomal regions that seem to be linked to specific disease phenotypes or partial phenotypes. This is followed by identification of a gene or genes that underlie the region of linkage and detection of single-nucleotide polymorphisms (SNPs) within these (candidate) genes that may predispose to disease. SNPs are used in populations and family-based association studies to strengthen the confidence that a specific gene is causally related to the disease or partial phenotype. Many genome screens for atopy and atopic disorders have been completed, with multiple regions of the genome being linked to varying phenotypes in different population cohorts, reflecting the heterogeneity and complexity of this condition.