Iain Kilty

Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-κB activation and proinflammatory cytokine release in alveolar epithelial cells

Oxidative stress is implicated in lung inflammation due to its effect on proinflammatory gene transcription. Changes in gene transcription depend on chromatin remodeling and the relative activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Alterations in the nuclear histone acetylation:deacetylation balance may result in uncontrolled transcription of specific proinflammatory genes. We studied the effect of hydrogen peroxide (H2O2) and cigarette smoke condensate (CSC) on histone acetylation:deacetylation in human alveolar epithelial cells (A549). H2O2 and CSC significantly increased acetylation of histone H4 proteins and were associated with decreased HDAC activity and HDAC2 levels in A549 cells. Also, the decreased HDAC2 activity was due to protein modification by aldehydes and nitric oxide products. Pretreatment of A549 cells with N‐acetyl‐L‐cysteine attenuated the oxidant‐mediated reduction in HDAC activity. Treatment of A549 cells with CSC did not cause nuclear factor‐κB (NF‐κB) activation or expression and release of either interleukin (IL)‐8 or IL‐6. However, H2O2, tumor necrosis factor‐α (TNF‐α), and IL‐1β significantly increased NF‐κB activation and expression of IL‐8 compared with control cells. Interestingly, CSC dose dependently inhibited TNF‐α‐ and IL‐ 1β‐mediated NF‐κB activation and IL‐8 expression. Thus, H2O2 and CSC enhance acetylation of histone proteins and decrease histone deacetylase activity but differentially regulate proinflammatory cytokine release in alveolar epithelial cells.

Improved image analysis workflow for 2-D gels enables large-scale 2-D gel-based proteomics studies--COPD biomarker discovery study.

2‐D gel electrophoresis has been used for more than three decades to study the protein complement of organisms, tissues, and cells. Three issues are holding back large‐scale proteomics studies: low‐throughput, high technical variation, and study designs lacking statistical power. We identified image analysis as the central factor connecting these three issues. By developing an improved image analysis workflow we shortened project timelines, decreased technical variation, and thus enabled large‐scale proteomics studies that are statistically powered. Rather than detecting protein spots on each gel image and matching spots across gel images, the improved workflow is based on aligning images first, then creating a consensus spot pattern and finally propagating the consensus spot pattern to all gel images for quantitation. This results in a data table without gaps. As an example we show here a study aimed at discovering circulating biomarkers for chronic obstructive pulmonary disease (COPD). Eight candidate biomarkers were identified by comparing plasma from 24 smokers with COPD and 24 smokers without COPD. Among the candidates are proteins such as plasma retinal‐binding protein (RETB) and fibrinogen that had previously been linked to the disease and are frequently monitored in COPD patients, as well as other proteins such as apolipoprotein E (ApoE), inter‐α‐trypsininhibitor heavy chain H4 (ITIH4), and glutathione peroxidase.

Regulation of 15-lipoxygenase isozymes and mucin secretion by cytokines in cultured normal human bronchial epithelial cells.

Abstract:Objective and Design: To study the effects of IL-4, IL-13, IL-1β, IL-8 and TNFα on 15-lipoxygenase(15-LO) isozyme expression and mucin secretion in normal human bronchial epithelial cells.¶Material and Methods: The effects of IL-4, IL-13, IL-1β, IL-8 and TNFα on 15-LO isoenzyme mRNA and protein expression, total 15-LO enzyme activity and mucin secretion were examined in cultures of normal human bronchial epithelial cells. In addition, in order to determine whether the observed effects on mucin secretion were due to lipoxygenase (LO) products, the effect of the non-selective LO inhibitor nordihydroguaiaretic acid was examined.¶Results: IL-4 and IL-13 selectively enhanced 15-LOa mRNA and protein levels, and total 15-LO enzyme activity. In contrast, no changes were observed in 15-LOb mRNA or protein levels. IL-4 and IL-13 both reduced mucin secretion in this cell type, however the non-selective LO inhibitor nordihydroguaiaretic acid had no effect on this action of IL-4.¶Conclusions: These data demonstrate that IL-4 and IL-13 selectively regulate the expression of the 15-LOa isozyme. However, 15LOa products do not mediate the IL-4-induced reduction in mucin secretion observed in this cell type.

Fragment based discovery of a novel and selective PI3 kinase inhibitor

We report the use of fragment screening and fragment based drug design to develop a PI3γ kinase fragment hit into a lead. Initial fragment hits were discovered by high concentration biochemical screening, followed by a round of virtual screening to identify additional ligand efficient fragments. These were developed into potent and ligand efficient lead compounds using structure guided fragment growing and merging strategies. This led to a potent, selective, and cell permeable PI3γ kinase inhibitor with good metabolic stability that was useful as a preclinical tool compound.

LPS challenge in healthy subjects: An investigation of neutrophil chemotaxis mechanisms involving CXCR1 and CXCR2

LPS inhalation was used to investigate whether sputum supernatant post-LPS challenge increases neutrophil chemotactic activity and to elucidate the role of CXCR1/CXCR2 signalling in this process. 14 healthy non-smoking subjects inhaled 30μg of LPS. Sputum was induced at baseline, 6 and 24h post-LPS challenge. Differential cell counts were determined and supernatants CXCL8, CXCL1, IL-6 and CCL2 levels measured. Peripheral blood neutrophils obtained from healthy volunteers were used for chemotaxis experiments using sputum supernatant. To delineate signalling mechanisms, the effects of a CXCR2/CXCR1 (dual) antagonist (Sch527123) and a CXCR2 specific antagonist (SB656933) were tested. LPS inhalation significantly increased sputum neutrophil counts from 45.3% to 76.7% and 69.3% at 6 and 24h respectively. LPS increased CXCL8, IL-6 and CCL2 levels but not CXCL1. Neutrophil chemotaxis significantly increased (2.7 fold) at 24h compared to baseline. Chemotaxis was inhibited by 79.0% with Sch527123 and 52.0% with SB656933. We conclude that LPS challenge increases sputum supernatant CXCL8 levels, which is associated with increased chemotactic activity which is dependent on both CXCR1 and CXCR2.

Chronic Obstructive Pulmonary Disease-Specific Gene Expression Signatures of Alveolar Macrophages as well as Peripheral Blood Monocytes Overlap and Correlate with Lung Function

Background: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by progressive airflow limitation and significant extrapulmonary (systemic) effects that lead to co-morbid conditions, though the pathomechanism of COPD is largely undetermined. Alveolar macrophages (AM) derived from peripheral monocytes (MO) appear to play a key role in initiating and/or sustaining disease progression. Objectives: To identify disease- and cell type-specific gene expression profiles and potential overlaps in those in order to diagnose COPD, characterize its progression and determine the effect of drug treatment. Method: Global gene expression analysis was used for primary screening in order to obtain expression signatures of AMs and circulating MOs of COPD patients and healthy controls. The results of microarray analyses of AMs (20 controls and 26 COPD patients) and MOs (16 controls and 22 COPD patients) were confirmed and validated by real-time quantitative polymerase chain reaction. Results: We have identified gene sets specifically associated with COPD in AMs and MOs. There were overlapping genes between the two cell types. Our data also show that COPD-specific gene expression signatures in AMs and MOs correlate with percent of predicted FEV1. Conclusion: Disease-specific and overlapping gene expression signatures can be defined in lung-derived macrophages and also in circulating monocytes. Some of the validated expression changes in both cell types correlate with lung function and therefore could serve as biomarkers of disease progression.

Effects of multikinase inhibitors on pressure overload-induced right ventricular remodeling

BACKGROUND Little is known about the effects of current PAH therapies and receptor tyrosine kinase inhibitors on heart remodeling. We sought to investigate the effects of the multikinase inhibitors sunitinib (PDGFR-, VEGFR- and KIT-inhibitor) and sorafenib (raf1/b-, VEGFR-, PDGFR-inhibitor) on pressure overload induced right ventricular (RV) remodeling. METHODS We investigated the effects of the kinase inhibitors on hemodynamics and remodeling in rats subjected either to monocrotaline (MCT)-induced PH or to surgical pulmonary artery banding (PAB). MCT rats were treated from days 21 to 35 with either vehicle, sunitinib (1mg/kg, 5mg/kg and 10mg/kg/day) or sorafenib (10mg/kg/day). PAB rats were treated with vehicle, sunitinib (10mg/kg/day) or sorafenib (10mg/kg/day) from days 7 to 21. RV function and remodeling were determined using echocardiography, invasive hemodynamic measurement and histomorphometry. RESULTS Treatment with both sorafenib and sunitinib decreased right ventricular systolic pressure, pulmonary vascular remodeling, RV hypertrophy and fibrosis in MCT rats. This was associated with an improvement of RV function. Importantly, after PAB, both compounds reversed RV chamber and cellular hypertrophy, reduced RV interstitial and perivascular fibrosis, and improved RV function. CONCLUSION We demonstrated that sunitinib and sorafenib reversed RV remodeling and significantly improved RV function measured via a range of invasive and non-invasive cardiopulmonary endpoints in experimental models of RV hypertrophy.

The role of IFN-γ in regulation of IFN-γ-inducible protein 10 (IP-10) expression in lung epithelial cell and peripheral blood mononuclear cell co-cultures

BackgroundInterferons play a critical role in regulating both the innate and adaptive immune responses. Previous reports have shown increased levels of IFN-γ, IFN-γ-inducing IL-12 and IFN-γ-inducible chemokine IP-10 in patients with chronic obstructive pulmonary disease (COPD).MethodsThe present study focuses on the regulation of the IP-10 secretion in co-cultures of lung epithelial cells and peripheral blood mononuclear cells (PBMCs).ResultsNo IP-10 secretion was detected in cells cultured alone, whereas a significant increase in IP-10 levels was observed in epithelial cell/PBMC co-cultures. Furthermore, the results show that interactions between lung epithelial cells, lymphocytes and monocytes are needed for basal IP-10 secretion. Interestingly, we have also shown that incubation with IL-12 can induce an IFN-γ independent increase in IP-10 levels in co-cultures. Furthermore, inhibition studies supported the suggestion that different intracellular pathways are responsible of IFN-γ and IL-12 mediated IP-10 secretion.ConclusionThese studies demonstrate a novel diversity in IFN-γ/IL-12 pathways, showing that the IP-10 expression in co-cultures is regulated by multiple factors, such as intercellular interactions in addition to IFN-γ and IL-12 levels. These results may be valuable in designing novel strategies to antagonize IP-10 mediated immunological reactions and chemotactic effects on T cells.

Cigarette Smoke Induced Airway Inflammation Is Independent of NF-κB Signalling

Rationale COPD is an inflammatory lung disease largely associated with exposure to cigarette smoke (CS). The mechanism by which CS leads to the pathogenesis of COPD is currently unclear; it is known however that many of the inflammatory mediators present in the COPD lung can be produced via the actions of the transcription factor Nuclear Factor-kappaB (NF-κB) and its upstream signalling kinase, Inhibitor of κB kinase-2 (IKK-2). Therefore the NF-κB/IKK-2 signalling pathway may represent a therapeutic target to attenuate the inflammation associated with COPD. Aim To use a range of assays, genetically modified animals and pharmacological tools to determine the role of NF-κB in CS-induced airway inflammation. Methods NF-κB pathway activation was measured in pre-clinical models of CS-induced airway inflammation and in human lung tissue from COPD patients. This data was complemented by employing mice missing a functional NF-κB pathway in specific cell types (epithelial and myeloid cells) and with systemic inhibitors of IKK-2. Results We showed in an airway inflammation model known to be NF-κB-dependent that the NF-κB pathway activity assays and modulators were functional in the mouse lung. Then, using the same methods, we demonstrated that the NF-κB pathway appears not to play an important role in the inflammation observed after exposure to CS. Furthermore, assaying human lung tissue revealed that in the clinical samples there was also no increase in NF-κB pathway activation in the COPD lung, suggesting that our pre-clinical data is translational to human disease. Conclusions In this study we present compelling evidence that the IKK-2/NF-κB signalling pathway does not play a prominent role in the inflammatory response to CS exposure and that this pathway may not be important in COPD pathogenesis.

Discovery of Clinical Candidate 1-{[(2S,3S,4S)-3-Ethyl-4-fluoro-5-oxopyrrolidin-2-yl]methoxy}-7-methoxyisoquinoline-6-carboxamide (PF-06650833), a Potent, Selective Inhibitor of Interleukin-1 Receptor Associated Kinase 4 (IRAK4), by Fragment-Based Drug Design

Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound 40). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.