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Interleukin (IL)-11 was originally recognized as an immunomodulatory and hematopoiesis-inducing cytokine. However, although IL-11 is typically not found in healthy individuals, it is now becoming evident that IL-11 may play a role in diverse pulmonary conditions, including IPF, asthma, and lung cancer. Additionally, experimental strategies targeting IL-11, such as humanized antibodies, have recently been developed, revealing the therapeutic potential of IL-11. Thus, further insight into the underlying mechanisms of IL-11 in lung disease may lead to the ability to interfere with pathological conditions that have a clear need for disease-modifying treatments, such as IPF. In this review, we outline the effects, expression, signaling, and crosstalk of IL-11 and focus on its role in lung disease and its potential as a therapeutic target.

SYK has been reported to possess both tumour promotor and repressor activities and deletion has been linked to a pro-proliferative / pro-invasive phenotype in breast tumours. It is unclear whether this is a consequence of protein deletion or loss of kinase activity. The SYK inhibitor, BI 1002494, caused no increase in proliferation in breast cancer cells or primary mammary epithelial cells in 2D or 3D cultures, nor changes in proliferation (CD1/2, CDK4, PCNA, Ki67) or invadopodia markers (MMP14, PARP, phospho-vimentin Ser56). BI 1002494 did not alter SYK protein expression. There was no change in phenotype observed in 3D cultures after addition of BI 1002494. Thirteen weeks of treatment with BI 1002494 resulted in no ductal branching or cellular proliferation in the mammary glands of mice. An in silico genetic analysis in breast tumour samples revealed no evidence that SYK has a typical tumour suppressor gene profile such as focal deletion, inactivating mutations or lower expression levels. Furthermore, SYK mutations were not associated with reduction in survival and disease-free period in breast cancer patients. In conclusion, small molecule inhibition of the kinase function of SYK does not contribute to a typical tumour suppressor profile.

Introduction: Cough is a debilitating symptom with a significant impact on quality of life. Patients with refractory chronic cough typically cough hundreds of times per day therefore we hypothesised that the associated pressures and shearing forces may lead to micro-injury of the pulmonary tissues. Aims: To compare lung and tissue injury biomarkers in the blood of refractory chronic cough patients (RCC) with healthy volunteers (HV). Methods: Serum from 64 RCC (75% female; mean age 54.3yrs) and 34 HV (62% female; mean age 36.7yrs) was assayed. Surfactant Protein-D (SP-D) and matrix metalloproteinase (MMP)-7 were measured using a commercial ELISA kit; C Reactive protein (CRP), Intercellular Adhesion Molecule (ICAM)-1, Serum amyloid A (SAA), Vascular cell adhesion protein (VCAM)-1 were measured using V-PLEX Vascular Injury Panel 2. Two tailed Mann-Whitney-U tests were performed. Results: All markers of micro-injury were significantly elevated in RCC patients compared with HV, with the most striking elevations seen in SP-D (302 vs 187 ng/mL, p Conclusion: Markers of epithelial damage (SP-D and MMP-7), vascular injury (VCAM-1 and ICAM-1) and peripheral tissue injury (CRP and SAA) were all elevated in the serum of chronic cough patients, supporting the hypothesis that cough is associated with lung micro-injury.

Unveiling the molecular mechanisms of tissue remodelling following injury is imperative to elucidate its regenerative capacity and aberrant repair in disease. Using different omics approaches, we identified enhancer of zester homolog 2 (EZH2) as a key regulator of fibrosis in injured lung epithelium. Epithelial injury drives an enrichment of nuclear transforming growth factor‐β‐activated kinase 1 (TAK1) that mediates EZH2 phosphorylation to facilitate its liberation from polycomb repressive complex 2 (PRC2). This process results in the establishment of a transcriptional complex of EZH2, RNA‐polymerase II (POL2) and nuclear actin, which orchestrates aberrant epithelial repair programmes. The liberation of EZH2 from PRC2 is accompanied by an EZH2‐EZH1 switch to preserve H3K27me3 deposition at non‐target genes. Loss of epithelial TAK1, EZH2 or blocking nuclear actin influx attenuates the fibrotic cascade and restores respiratory homeostasis. Accordingly, EZH2 inhibition significantly improves outcomes in a pulmonary fibrosis mouse model. Our results reveal an important non‐canonical function of EZH2, paving the way for new therapeutic interventions in fibrotic lung diseases., TAK1‐mediated phosphorylation of EZH2 regulates fibrotic epithelial remodelling after injury. EZH2 inhibition improves outcomes in a pulmonary fibrosis model, suggesting that targeting the TAK1‐EZH2 axis has therapeutic efficacy for idiopathic pulmonary fibrosis (IPF).

Background & aimRecurring epithelial injury and aberrant repair are considered as a major driver of idiopathic pulmonary fibrosis (IPF) leading to chronic inflammation, fibroblast activation and ultimately to scarring and stiffening of the lung. As decline of lung function is the first reported symptom by IPF patients and occurs once fibrosis is firmly established, animal models are required to study early disease-driving mechanisms.MethodsWe developed a novel and flexible mouse model of acute epithelial injury based on adeno-associated virus (AAV) variant 6.2 mediated expression of the human diphtheria toxin receptor (DTR). Following intratracheal administration of diphtheria toxin (DT), a cell-specific death of bronchial epithelial and alveolar epithelial type II cells can be observed.ResultsDetailed characterization of the AAV-DTR/DT mouse model revealed increasing cell numbers in bronchoalveolar lavage (BAL; macrophages, neutrophils, and atypical cells) and elevation of apoptotic cells and infiltrated leukocytes in lung tissue, which were dependent of viral genome load and DT dose. Cytokine levels in BAL fluid showed different patterns dependent of viral genome load with IFNγ, TNFα, and IP-10 increasing and IL-5 and IL-6 decreasing, while lung function was not affected. Additionally, laser-capture microdissection-based proteomics of bronchial and alveolar epithelium showed upregulated immune and inflammatory response in all epithelial cell regions and extracellular matrix deposition in infiltrated alveoli, while proteins involved in pulmonary surfactant synthesis, alveolar fluid clearance and alveolar-capillary barrier were downregulated in the parenchyma.ConclusionOur novel AAV-DTR/DT model resembles specific aspects of pulmonary diseases like IPF and acute respiratory distress syndrome.Short summary for social mediaA novel and flexible mouse model of acute epithelial lung injury based on AAV-mediated expression of the human diphtheria toxin receptor followed by intratracheal instillation of diphtheria toxin resembles specific aspects of pulmonary diseases like IPF.

In fibroblasts, TGFβ1 stimulates IL11 upregulation that leads to an autocrine loop of IL11-dependent pro-fibrotic protein translation. The signalling pathways downstream of IL11 are contentious and both STAT3 and ERK have been implicated. Here we show that TGFβ1- or IL11-induced ERK activation is consistently associated with fibrogenesis whereas STAT3 phosphorylation (pSTAT3) is unrelated to fibroblast activation. Surprisingly, recombinant human IL11, which has been used extensively in mouse experiments to infer STAT3 activity downstream of IL11, non-specifically increases pSTAT3 in Il11ra1 null mouse fibroblasts. Pharmacologic inhibition of STAT3 prevents TGFβ1-induced fibrogenesis but this effect was found to reflect fibroblast dysfunction due to severe proteotoxic ER stress. In contrast, inhibition of MEK/ERK prevented fibrosis in the absence of ER stress. TGFβ1-stimulated ERK/mTOR/P70RSK-driven protein translation was IL11-dependent and selectivity for pro-fibrotic protein synthesis was ascribed to an EPRS-related mechanism. In TGFβ1-stimulated fibroblasts, the anti-fibrotic drug nintedanib caused dose-dependent ER stress, reduced pSTAT/pERK and inhibited pro-fibrotic protein translation, similarly to generic STAT3 inhibitors or ER stressors. Pirfenidone, while anti-fibrotic, had no effect on ER stress whereas anti-IL11 inhibited the ERK/mTOR axis while reducing ER stress. These studies discount a specific role for STAT3 in pro-fibrotic signaling, suggest a novel mechanism of action for nintedanib and prioritise further the IL11 pathway as a therapeutic target for fibrosis.

Background Asthma is a chronic respiratory disease in which the nervous system plays a central role. Sensory nerve activation, amongst others via Transient Receptor Potential Ankyrin 1 (TRPA1) channels, contributes to asthma characteristics including cough, bronchoconstriction, mucus secretion, airway hyperresponsiveness (AHR) and inflammation. In the current study, we evaluated the efficacy of the novel TRPA1 antagonist BI01305834 against AHR and inflammation in guinea-pig models of asthma. Methods First, a pilot study was performed in a guinea-pig model of allergic asthma to find the optimal dose of BI01305834. Next, the effect of BI01305834 on (1) AHR to inhaled histamine after the early and late asthmatic reaction (EAR and LAR), (2) magnitude of EAR and LAR and (3) airway inflammation was assessed. Precision-cut lung slices and trachea strips were used to investigate the bronchoprotective and bronchodilating-effect of BI01305834. Statistical evaluation of differences of in vivo data was performed using a Mann–Whitney U test or One-way nonparametric Kruskal–Wallis ANOVA, for ex vivo data One- or Two-way ANOVA was used, all with Dunnett’s post-hoc test where appropriate. Results A dose of 1 mg/kg BI01305834 was selected based on AHR and exposure data in blood samples from the pilot study. In the subsequent study, 1 mg/kg BI01305834 inhibited AHR after the EAR, and the development of EAR and LAR elicited by ovalbumin in ovalbumin-sensitized guinea pigs. BI01305834 did not inhibit allergen-induced total and differential cells in the lavage fluid and interleukin-13 gene expression in lung homogenates. Furthermore, BI01305834 was able to inhibit allergen and histamine-induced airway narrowing in guinea-pig lung slices, without affecting histamine release, and reverse allergen-induced bronchoconstriction in guinea-pig trachea strips. Conclusions TRPA1 inhibition protects against AHR and the EAR and LAR in vivo and allergen and histamine-induced airway narrowing ex vivo, and reverses allergen-induced bronchoconstriction independently of inflammation. This effect was partially dependent upon histamine, suggesting a neuronal and possible non-neuronal role for TRPA1 in allergen-induced bronchoconstriction.

Background: Spleen tyrosine kinase (SYK) inhibitors have therapeutic potential for severe asthma. In rats, activity of SYK inhibitor BI 1002494 measured with a CD63 assay correlated well with bronchoconstriction and airway resistance (Lamb DJ, et al. JPET 2016; 357:554–61). Aim: To evaluate a CD63 assay as a biomarker for SYK inhibitor activity in humans. Methods: Healthy volunteer whole blood was sensitized with anti-dinitroprusside (DNP) immunoglobulin E (IgE) and challenged with DNP. SYK inhibitor activity was measured with surface expression of CD63 as an index of basophil degranulation. Results: Strong correlation between CD63 IC50 and histamine release IC50 (R2=0.998) across 14 SYK inhibitor compounds confirmed CD63 as a good surrogate for functional basophil degranulation. BI 1002494 had a median CD63 IC50 of 113 nM (95% CI 103, 135) across 153 consecutive assays, with inter-sample variability of 17–2530 nM. Individual concentration curve gradients for basophil activation by the SYK inhibitor did not correlate with IC50 (R2=0.15). The proportion of basophils activated by anti-DNP/DNP (14–100%) correlated with IC50 (R2=0.44), suggesting that the more IgE receptors (i.e. the more SYK molecules) engaged, the greater the amount of inhibitor required. Similar assay performance and IC50 values from clinical biomarker labs indicate that the assay is robust and translatable. For 1000 SYK inhibitors, poor correlation was found between enzymatic kinase activity and CD63 potency (R2=0.22), possibly due to differences in cellular permeability and plasma protein binding. Conclusion: Employing a CD63 assay in ex vivo stimulated human whole blood could be used for screening compounds for human SYK inhibitor activity.

Smoking is an important risk factor for the development of chronic obstructive pulmonary disease (COPD) and viral infections are believed to be major triggers of exacerbations, which periodically lead to a worsening of symptoms. The pro-inflammatory IL-1 family members IL-1α and IL-1β are increased in COPD patients and might contribute to disease pathology. We investigated whether individual or combined inhibition of these cytokines reduced lung inflammation in cigarette smoke (CS)-exposed and H1N1-infected BALB/c mice. Animals were treated with individual or combined antibodies (Abs) directed against IL-1α, IL-1β or IL-1R1. Cells in BAL fluid and cytokines/chemokines in lung homogenate were determined. The viral load was investigated. Blocking IL-1α had significant suppressive effects on total cells, neutrophils, and macrophages. Furthermore, it reduced KC levels significantly. Blocking of IL-1β did not provide significant activity. In primary human bronchial epithelial air-liquid-interface cell cultures infected with H1N1, IL-1α Abs but not IL-1β Abs reduced levels of TNF-α and IL-6. Concomitant usage of Abs against IL-1α/IL-1β revealed strong effects in vivo and reduced total cells, neutrophils and macrophages. Additionally, levels of KC, IL-6, TNF-α, MCP-1, MIP-1α and MIP-1β were significantly reduced and ICAM-1 and MUC5 A/C mRNA expression was attenuated. The viral load decreased significantly upon combined IL-1α/IL-1β Ab treatment. Blocking the IL-1R1 provided significant effects on total cells, neutrophils and macrophages but was inferior compared to inhibiting both its soluble ligands IL-1α/IL-1β. Our results suggest that combined inhibition of IL-1α/IL-1β might be beneficial to reduce CS/H1N1-induced airway inflammation. Moreover, combined targeting of both IL-1α/IL-1β might be more efficient compared to individual neutralization IL-1α or IL-1β or inhibition of the IL-1R1.

In addition to uPA’s well-characterized role in fibrinolysis, recent publications have indicated a role for uPA-uPAR signaling in respiratory epithelia barrier function. Furthermore, uPA and uPAR expression/signaling are enhanced in severe asthmatic and COPD airway epithelium. We therefore hypothesised that uPA-uPAR signaling promotes epithelial barrier dysfunction and airway remodeling in severe asthma and COPD. We showed that exogenous addition of uPA to primary airway basal cells during differentiation resulted in reduced epithelial thickness and remodeling. Exposure of fully differentiated airway cultures to various damage stimuli (TNFα, IL-1β, LPS, PolyIC) induced endogenous uPA secretion. Supernatant uPA concentrations directly correlated with epithelial barrier breakdown (P = 0.01), as measured by TEER and dextran permeability. Treatment of fully differentiated airway epithelial cultures from healthy, severe asthmatic and COPD donors, with an uPA-uPAR inhibitor (IPR-1110; Liu et al. ACSChem.Biol.2015,10,1521-1534) attenuated TNFα-induced increases in epithelial permeability. Moreover, addition of IPR-1110 72hours post TNFα-induced epithelial damage restored epithelial permeability by ~55% (relative to TNFα only control). Preliminary data indicates that IPR-1110 reduced the phosphorylation of kinases previously shown to be linked to uPA-uPAR signaling, including Erk, Akt and p38. Screening of epithelial tight/adherens junction proteins, revealed that IPR-1110 induced changes in epithelial barrier function correlated with enhanced expression of JAM-A. Thus, targeting uPA-uPAR signaling has therapeutic potential to restore epithelial barrier function in severe asthma and COPD.

The above article from the British Journal of Pharmacology, published online on May 20, 2020 in Wiley Online Library (http://wileyonlinelibrary.com) has been withdrawn due to a lack of full disclosure of the chemical structure of the novel TRPA1 antagonist BI01305834, by agreement between the Editor-in-Chief and John Wiley & Sons Inc on behalf of The British Pharmacology Society.

Transient receptor potential vanilloid 4 (TRPV4) has been implicated in many disease conditions also in the lung. Its activation leads to an increase endothelial permeability in an intracellular calcium-influx dependent manner. We investigated its function in vitro on primary human endothelial cells using two TRPV4 agonists, GSK1016790A and 4α-Phorbol 12,13-didecanoate (4α-PDD) and a selective TRPV4 blocker GSK2193874. Both TRPV4 agonists leaded to a reduction in transendothelial electrical resistance (TER) which was mediated however by differential cytotoxic effects. 4α-PDD induced apoptosis that could not be blocked by TRPV4 inhibition in HUVECs, whereas GSK1016790A selectively activated TRPV4 and reduced TER as a consequence of cellular necrosis. TRPV4 mediated cytotoxicity is poorly described and may provide significant context to the role of TRPV4 in barrier-function., Highlights • TRPV4 agonism is widely associated with barrier-dysfunction. • We show TRPV4 mediated increased membrane permeability is caused by cytotoxicity. • The TRPV4 agonist GSK1016790A mediates barrier dysfunction via necrosis. • The TRPV4 agonist 4α-PDD mediates barrier dysfunction via apoptosis.

Steroid refractory inflammation is an unmet medical need in the management of inflammatory diseases. Thus, mechanisms, improving steroid sensitivity and simultaneously decreasing inflammation have potential therapeutic utility. The FK506‐binding protein 51 (FKBP51) is reported to influence steroid sensitivity in mental disorders. Moreover, biochemical data highlight a connection between FKBP51 and the IKK complex. The aim of this study was to elucidate whether FKBP51 inhibition had utility in modulating steroid resistant inflammation by increasing the sensitivity of the glucocorticoid receptor (GR) signalling and simultaneously inhibiting NFκB‐driven inflammation. We have demonstrated that FKBP51 silencing in a bronchial epithelial cell line resulted in a 10‐fold increased potency for dexamethasone towards IL1beta‐induced IL6 and IL8, whilst FKBP51 over‐expression of FKBP51 reduced significantly the prednisolone sensitivity in a murine HDM‐driven pulmonary inflammation model. Immunoprecipitation experiments with anti‐FKBP51 antibodies, confirmed the presence of FKBP51 in a complex comprising Hsp90, GR and members of the IKK family. FKBP51 silencing reduced NFκB (p50/p65) nucleus translocation, resulting in reduced ICAM expression, cytokine and chemokine secretion. In conclusion, we demonstrate that FKBP51 has the potential to control inflammation in steroid insensitive patients in a steroid‐dependent and independent manner and thus may be worthy of further study as a drug target.

Mechanical ventilation is an important tool for supporting critically ill patients but may also exert pathological forces on lung cells leading to Ventilator-Induced Lung Injury (VILI). We hypothesised that inhibition of the force-sensitive transient receptor potential vanilloid (TRPV4) ion channel may attenuate the negative effects of mechanical ventilation. Mechanical stretch increased intracellular Ca2+ influx and induced release of pro-inflammatory cytokines in lung epithelial cells that was partially blocked by about 30% with the selective TRPV4 inhibitor GSK2193874, but nearly completely blocked with the pan-calcium channel blocker ruthenium red, suggesting the involvement of more than one calcium channel in the response to mechanical stress. Mechanical stretch also induced the release of pro-inflammatory cytokines from M1 macrophages, but in contrast this was entirely dependent upon TRPV4. In a murine ventilation model, TRPV4 inhibition attenuated both pulmonary barrier permeability increase and pro-inflammatory cytokines release due to high tidal volume ventilation. Taken together, these data suggest TRPV4 inhibitors may have utility as a prophylactic pharmacological treatment to improve the negative pathological stretch-response of lung cells during ventilation and potentially support patients receiving mechanical ventilation.

Einleitung: Syk ist eine intrazellulare Nicht-Rezeptor-Tyrosinkinase, die jüngst als zentraler Mediator glattmuskulärer Konstriktion identifiziert wurde. Ob Syk eine funktionelle Rolle in der pulmonalen Vasokonstriktion ausübt und sich hieraus mögliche therapeutische Implikationen für die pulmonalarterielle Hypertonie (PAH) ableiten lassen, ist gegenwärtig unbekannt. Methoden: Das vaskuläre Expressionsprofil von Syk wurde sowohl in humanem (PAH versus Nicht-PAH) als auch in murinem Lungengewebe mittels Konfokal-Mikroskopie charakterisiert. Die funktionelle Rolle von Syk in der pulmonalen Vasokonstriktion wurde mittels Einsatz von Syk- und/oder NO-Inhibitoren in humanen Precision-Cut Lung Slices (PCLS) und in isoliert perfundierten und ventilierten Lungen von Wildtyp-Mäusen oder von eNOS- oder PKCa-defizienten Mäusen untersucht. Die Effekte der Syk-Inhibition auf die PAH-assoziierte pulmonalvaskuläre Hyperreagibilität wurden in isolierten Mauslungen nach Induktion pulmonaler TH2-Inflammation analysiert. Ergebnisse: Syk ist sowohl beim Menschen als auch in der Maus in den glatten Gefäßmuskelzellen der Lunge exprimiert. Die Inhibition von Syk hatte eine deutliche Reduktion der Serotonin-, Endothelin-1- und Angiotensin II-induzierten pulmonalen Vasokonstriktion in humanen PCLS und/oder in isoliert perfundierten Mauslungen zur Folge, unabhängig von eNOS oder PKCa. In präkonstringierten Pulmonalgefäßen führte Syk-Inhibition NO-unabhängig zu einer raschen, reversiblen Vasodilatation. Darüber hinaus reduzierte Syk-Inhibition sowohl die hypoxisch pulmonale Vasokonstriktion als auch die TH2-induzierte pulmonalvaskuläre Hyperreagibilität. Diskussion: Syk reguliert die pulmonale Vasokonstriktion unabhängig von NO. Unsere Daten implizieren, dass Syk-Inhibition eine innovative Therapieform in der Behandlung der PAH darstellen könnte.

Introduction: In the airways, spleen tyrosine kinase (Syk) promotes inflammation, smooth muscle cell proliferation and contraction (Tabeling, C. et al. Allergy 2017 Jul;72(7):1061-1072). However, little is known about the expression and role of Syk in the vascular compartment of the lung. Here, we analyzed Syk expression and function in the pulmonary vasculature and its possible involvement in the pathogenesis of pulmonary arterial hypertension (PAH). Methods: In human (PAH vs. donor) and murine lungs, Syk expression was assessed by immunofluorescence and spectral confocal microscopy. Syk function was analyzed in human precision-cut lung slices (PCLS) and in isolated perfused lungs of wild-type mice or mice deficient in eNOS, PKCα or mast cells with or without inhibition of Syk, PKC, rho kinase, p38 MAPK and/or NO synthase. Pulmonary vascular hyperresponsiveness was investigated following induction of pulmonary Th2 inflammation. Results: Syk was expressed in pulmonary arterial smooth muscle cells of both control and PAH lungs. Syk inhibition (either with BAY 61-3606 or with BI 1002494) reduced pulmonary vasoconstriction in human PCLS and in isolated mouse lungs independent of eNOS, PKCα or mast cells. In preconstricted lungs, Syk inhibition rapidly reversed vasoconstriction in a NO-independent manner. Pulmonary vascular hyperresponsiveness was markedly diminished following Syk inhibition. Inhibition of p38 MAPK reduced pulmonary vasoconstriction to the same extent as Syk inhibition, and simultaneous inhibition of p38 MAPK and Syk had no additive inhibitory effect when compared to Syk inhibition only. Conclusions: Syk regulates pulmonary vasoconstriction, presumably via p38 MAPK, and may be a promising target in PAH therapy.

Inappropriate repair responses to pulmonary epithelial injury have been linked to perturbation of epithelial barrier function and airway remodelling in a number of respiratory diseases, including chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. We developed an in vitro mechanical scratch injury model in air-liquid interface differentiated primary human small airway epithelial cells that recapitulates many of the characteristics observed during epithelial wound injury in both human tissue and small animal models. Wound closure was initially associated with de-differentiation of the differentiated apical cells and rapid migration into the wound site, followed by proliferation of apical cells behind the wound edge, together with increases in FAK expression, fibronectin and reduction in PAI-1 which collectively facilitate cell motility and extracellular matrix deposition. Macrophages are intimately involved in wound repair so we sought to investigate the role of macrophage sub-types on this process in a novel primary human co-culture model. M1 macrophages promoted FAK expression and both M1 and M2 macrophages promoted epithelial de-differentiation. Interestingly, M2a macrophages inhibited both proliferation and fibronectin expression, possibly via the retinoic acid pathway, whereas M2b and M2c macrophages prevented fibronectin deposition, possibly via MMP expression. Collectively these data highlight the complex nature of epithelial wound closure, the differential impact of macrophage sub-types on this process, and the heterogenic and non-delineated function of these macrophages. published

Background Spleen tyrosine kinase (Syk) is an intracellular non-receptor tyrosine kinase, which has been implicated as central immune modulator promoting allergic airway inflammation. Syk inhibition has been proposed as a new therapeutic approach in asthma. However, the direct effects of Syk inhibition on airway constriction independent of allergen sensitization remain elusive. Methods Spectral confocal microscopy of human and murine lung tissue was performed to localize Syk expression. The effects of prophylactic or therapeutic Syk inhibition on allergic airway inflammation, hyperresponsiveness and airway remodeling were analyzed in allergen-sensitized and airway-challenged mice. The effects of Syk inhibitors BAY 61-3606 or BI 1002494 on airway function were investigated in isolated lungs of wild-type, PKCα-deficient, mast cell-deficient or eNOS-deficient mice. Results Syk expression was found in human and murine airway smooth muscle cells. Syk inhibition reduced allergic airway inflammation, airway hyperresponsiveness and pulmonary collagen deposition. In naive mice, Syk inhibition diminished airway responsiveness independently of mast cells, or PKCα or eNOS expression and rapidly reversed established bronchoconstriction independently of NO. Simultaneous inhibition of Syk and PKC revealed additive dilatory effects, whereas combined inhibition of Syk and rho kinase or Syk and p38 MAPK did not cause additive bronchodilation. Conclusions Syk inhibition directly attenuates airway smooth muscle cell contraction independent of its protective immunomodulatory effects on allergic airway inflammation, hyperresponsiveness and airway remodeling. Syk mediates bronchoconstriction in a NO-independent manner, presumably via rho kinase and p38 MAPK, and Syk inhibition might present a promising therapeutic approach in chronic asthma as well as acute asthma attacks. This article is protected by copyright. All rights reserved.

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

Monocyte recruitment and their differentiation into macrophages are both early events in native and accelerated atherosclerosis that follows angioplasty. We have investigated the putative functional role of the epidermal growth factor receptor (EGFR) present on rabbit monocytes/macrophages. The impact of periadventitial delivery of an EGFR-specific, blocking monoclonal antibody (ICR62, which inhibits EGF-binding to its receptor) was investigated in a rabbit model of accelerated atherosclerosis induced by a combination of carotid injury and 4 weeks of a 2% cholesterol-diet. Two weeks after the initiation of the diet, a balloon-catheter angioplasty of the left common carotid artery was performed and a collar placed around the injured carotid artery immediately, for the delivery of ICR62 antibody, isotype-matched antibody or saline control. Monocyte/macrophage accumulation, cell proliferation and neointimal thickening were determined 2 weeks after the delivery of the antibodies. The function of the EGFR on rabbit monocytes was also investigated in vitro, using chemotaxis assays. Treatment with ICR62 was associated with a significant reduction in macrophage accumulation and neointimal thickening and a 76% reduction in neointimal area of the vessel wall compared with controls. In vitro ICR62 inhibited macrophage and smooth muscle cell migration towards EGFR ligands including EGF and HB-EGF. These findings suggest that EGFR ligation may be important in the development of early atherosclerotic lesions following balloon-catheter angioplasty, and periadventitial delivery may provide a feasible approach for administration of the inhibitors of EGFR-binding such as ICR62.

Asthma affects 300 million people worldwide and continues to be a major cause of morbidity and mortality. Disease relevant animal models of asthma are required for benchmarking of novel therapeutic mechanisms in comparison to established clinical approaches. We demonstrate that chronic exposure of mice to house dust mite (HDM) extract results in allergic airway inflammation, that can be significantly attenuated by therapeutic intervention with phosphodiesterase 4 inhibition and corticosteroid treatment. Female BALB/c mice were administered intranasally with HDM ( Dermatophagoides pteronyssinus ) extract daily for five weeks, and therapeutic intervention with anti-inflammatory treatment (dexamethasone 1 mg/kg subcutaneous once daily, prednisolone 10 mg/kg orally twice daily, fluticasone 3, 10 and 30 μg intranasally twice daily, roflumilast 10 mg/kg orally twice daily and intranasally 10 and 30 μg twice daily) was initiated after three weeks of exposure. Chronic HDM extract exposure resulted in significant airway inflammation, demonstrated by bronchoalveolar lavage cell infiltration and lung tissue inflammatory gene expression by TaqMan low density array. Chronic steroid treatment significantly inhibited these parameters. In addition, roflumilast caused a significant reduction in airway inflammatory cell infiltration. We have demonstrated that chronic HDM-induced allergic inflammation can be significantly ameliorated by steroid treatment, and that phosphodiesterase 4 inhibition modulates inflammatory cell infiltration. Therefore, the murine HDM model may be a useful tool for evaluating new targets for the treatment of asthma.

There has been considerable debate about how copper status may affect the biochemical and cellular processes associated with atherogenesis. We have investigated the effects of graded dietary copper supplementation on processes likely to contribute to atherogenesis, using the cholesterol-fed New Zealand White rabbit model. Rabbits (n = 40) were fed a 0.25-1% cholesterol diet deficient in copper. Animals received either 0, 1, 3 or 20 mg copper/day and were killed after 13 weeks. Plasma cholesterol levels were similar in each dietary group. Aortic concentrations of copper were higher in the 20 mg copper/day animals compared to those receiving 0 mg copper/day (3.70 +/- 0.78 vs. 1.33 +/- 0.46 microg/g wet tissue; P 0.05). En face staining of aortae with oil red O showed that both high copper supplementation (20 mg/day) (67.1 +/- 5.5%) and a deficient diet (0 mg/day) (63.1 +/- 4.8%) was associated with significantly larger lesions (P < 0.05) compared to moderately supplemented animals (1 mg/day and 3 mg/day) (51.3 +/- 6.3 and 42.8 +/- 7.9%). These data indicate that in the cholesterol-fed rabbit, there is an optimal dietary copper intake and that dietary copper deficiency or excess are associated with an increased susceptibility to aortic atherosclerosis. Many Western diets contain insufficient copper and these findings indicate that a moderate dietary copper content may confer a degree of cardiac protection to the human population.