David James Lamb

A Novel Model of IgE-Mediated Passive Pulmonary Anaphylaxis in Rats

Mast cells are central effector cells in allergic asthma and are augmented in the airways of asthma patients. Attenuating mast cell degranulation and with it the early asthmatic response is an important intervention point to inhibit bronchoconstriction, plasma exudation and tissue oedema formation. To validate the efficacy of novel pharmacological interventions, appropriate and practicable in vivo models reflecting mast cell-dependent mechanisms in the lung, are missing. Thus, we developed a novel model of passive pulmonary anaphylaxis in rats. Rats were passively sensitized by concurrent intratracheal and intradermal (ear) application of an anti-DNP IgE antibody. Intravenous application of the antigen, DNP-BSA in combination with Evans blue dye, led to mast cell degranulation in both tissues. Quantification of mast cell degranulation in the lung was determined by (1) mediator release into bronchoalveolar lavage, (2) extravasation of Evans blue dye into tracheal and bronchial lung tissue and (3) invasive measurement of antigen-induced bronchoconstriction. Quantification of mast cell degranulation in the ear was determined by extravasation of Evans blue dye into ear tissue. We pharmacologically validated our model using the SYK inhibitor Fostamatinib, the H1-receptor antagonist Desloratadine, the mast cell stabilizer disodium cromoglycate (DSCG) and the β2-adrenergic receptor agonist Formoterol. Fostamatinib was equally efficacious in lung and ear. Desloratadine effectively inhibited bronchoconstriction and ear vascular leakage, but was less effective against pulmonary vascular leakage, perhaps reflecting the differing roles for histamine receptor sub-types. DSCG attenuated both vascular leakage in the lung and bronchoconstriction, but with a very short duration of action. As an inhaled approach, Formoterol was more effective in the lung than in the ear. This model of passive pulmonary anaphylaxis provides a tissue relevant readout of early mast cell activity and pharmacological benchmarking broadly reflects responses observed in patients with asthma.

BI 1002494, a Novel Potent and Selective Oral Spleen Tyrosine Kinase Inhibitor, Displays Differential Potency in Human Basophils and B Cells

BI 1002494 [(R)-4-{(R)-1-[7-(3,4,5-trimethoxy-phenyl)-[1,6]napthyridin-5-yloxy]-ethyl}pyrrolidin-2-one] is a novel, potent, and selective spleen tyrosine kinase (SYK) inhibitor with sustained plasma exposure after oral administration in rats, which qualifies this molecule as a good in vitro and in vivo tool compound. BI 1002494 exhibits higher potency in inhibiting high-affinity IgE receptor–mediated mast cell and basophil degranulation (IC50 = 115 nM) compared with B-cell receptor–mediated activation of B cells (IC50 = 810 nM). This may be explained by lower kinase potency when the physiologic ligand B-cell linker was used, suggesting that SYK inhibitors may exhibit differential potency depending on the cell type and the respective signal transduction ligand. A 3-fold decrease in potency was observed in rat basophils (IC50 = 323 nM) compared with human basophils, but a similar species potency shift was not observed in B cells. The lower potency in rat basophils was confirmed in both ex vivo inhibition of bronchoconstriction in precision-cut rat lung slices and in reversal of anaphylaxis-driven airway resistance in rats. The different cellular potencies translated into different in vivo efficacy; full efficacy in a rat ovalbumin model (that contains an element of mast cell dependence) was achieved with a trough plasma concentration of 340 nM, whereas full efficacy in a rat collagen-induced arthritis model (that contains an element of B-cell dependence) was achieved with a trough plasma concentration of 1400 nM. Taken together, these data provide a platform from which different estimates of human efficacious exposures can be made according to the relevant cell type for the indication intended to be treated.

Opposing effects of in vitro differentiated macrophages sub-type on epithelial wound healing

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.

TRPV4 inhibition attenuates stretch-induced inflammatory cellular responses and lung barrier dysfunction during mechanical ventilation

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.

FKBP51 modulates steroid sensitivity and NFκB signalling: A novel anti-inflammatory drug target

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.

Characterisation of a Mouse Model of Cigarette Smoke Extract-Induced Lung Inflammation

Aim: The aim of this study was to develop a lung-targeted, mouse model of cigarette smoke-induced inflammation that can be used to study the pathophysiological changes that occur in the lungs of human smokers and patients with chronic obstructive pulmonary disease (COPD). Materials & methods: Cigarette smoke extract (CSE) was prepared freshly daily. Intranasal administration into female mice was performed once daily for up to 3 weeks. Results: CSE significantly increased airway macrophages after 3 and 4 days of dosing, and then declined over the subsequent 2 weeks. However, airway neutrophils were elevated after a single dose of CSE, and at all subsequent time points. Muc5AC was significantly increased in the Bronchoalveolar lavage (BAL) of CSE-treated animals compared to control mice (P<0.05), but TNF-α concentrations decreased in a dose-dependent manner. In animals challenged with CSE for 4 consecutive days, a PDE4 inhibitor (Roflumilast; 10 mg/kg BID) significantly inhibited both macrophages (P<0.01) and neutrophils (P<0.001), a steroid (prednisolone; 10 mg/kg BID) had no effect on either macrophages or neutrophils and an oral p38 inhibitor (PHA-818637; 10 mg/kg BID) inhibited macrophages (P<0.05), but not neutrophils. CSE inhibited lipopolysaccharide-induced airway neutrophilia. Conclusion: This model reflects many aspects of human COPD including pulmonary leucocytes, mucin, TNF-α and response to clinical therapeutic agents and may be useful in assessing the efficacy of potential therapies