Inhibitor of apoptosis proteins as regulatory factors in the normal and inflamed airways.

Asthma is potentiated by complex gene-environment interactions, and characterised by inflammation and degenerative changes to the conducting airways. Current therapeutics targeting the inflammation and bronchoconstriction are restricted to prophylactic effects. The airway epithelium is known to participate in the pathogenesis of asthma, and represents a therapeutic target. Airway epithelial cells (AEC) from asthmatics exhibit apoptotic changes, which correlate with disease-associated factors presented by the epithelium. However, the mechanisms which cause the apoptosis are not well defined. In particular, there has been little study of the role of the family of Inhibitor of Apoptosis Proteins (IAPs) in models of AEC apoptosis. The over-arching hypothesis in this thesis is that anomalies in one or more of the IAPs contribute to inflammation-induced AE apoptosis in asthma. Experiments in this thesis explored the role of XIAP, cIAP1 and cIAP2 in asthma, models of asthma-related inflammation, and genetic susceptibility to asthma. The major methods used in these experiments included cell culture of primary AEC from both asthmatics and controls with/without treatment with IFNγ and TNFα. siRNA knockdown, qPCR, western blotting, immunocytochemistry, functional caspase assays, and genotyping. The major findings of this study are i) surprisingly, primary AECs do not undergo apoptosis after prolonged exposure to the proinflammatory cytokines IFNγ and TNFα ex vivo; ii) rather, IFNγ elicits a proapoptotic state in AECs, evidenced by, partial processing of procaspase-3, the absence of Poly (ADP-ribose) polymerase (PARP) cleavage, an increased Bax:Bcl2 transcript ratio, and the absence of morphological changes associated with apoptosis; iii) both XIAP and cIAP1 were constitutively expressed in AEC, and protein levels were unaffected by cytokine treatment. In contrast cIAP2, initially weakly expressed, was strongly inducible by cytokine treatment; iv) No differences were observed between AEC from asthmatics and controls in terms of either basal IAP gene expression levels or their response to cytokine treatment; v) siRNA-mediated depletion of cIAP2- transcripts allows AEC to progress into apoptosis after extended culture, conditions which also resulted in a decrease in both cIAP1 and Bcl2; vi) genetic polymorphism in the genes encoding, XIAP cIAP1 and cIAP2 do not associate with susceptibility for asthma. However, cIAP1 polymorphism may modulate disease severity within asthmatics. This thesis contributes to the knowledge of IAPs and apoptosis in asthma, and provides evidence that they are important for sustaining AEC survival, and participate within a cooperative of endogenous regulators of apoptosis. There is no evidence of intrinsic dysregulation of IAPs in asthma, yet cIAP1 polymorphism may modulate asthma severity, and IAPs are central in maintaining a proapoptotic state in AECs exposed to asthma related cytokines. Epithelial activation and damage, coupled with non-progression to apoptosis may contribute to fragility of the AE observed in asthma. Therapies targeting the IAPs may therefore provide a means of ameliorating the disease by allowing AECs to progress into apoptosis.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 201

Dietary zinc mediates inflammation and protects against wasting and metabolic derangement caused by sustained cigarette smoke exposure in mice

In mouse asthma models, inflammation can be modulated by zinc (Zn). Given that appetite loss, muscle wasting and poor nutrition are features of chronic obstructive pulmonary disease (COPD) and that poor dietary Zn intake is in itself accompanied by growth retardation and appetite loss, we hypothesised that dietary Zn limitation would not only worsen airway inflammation but also exaggerate metabolic effects of cigarette smoke (CS) exposure in mice. Conversely, Zn supplementation would lessen inflammation. Mice were exposed to CS [2× 2RF, 3×/day; 15 min/cigarette] and fed diets containing 2, 20 or 140 mg/kg Zn ad libitum. Airway cells were collected by bronchoalveolar lavage (BAL). Plasma Zn was measured by fluorometric assay. Inflammatory, metabolic and Zn transport markers were measured by real-time RT-PCR. Mice fed low Zn diets had less plasma labile zinc (0-0.18 μM) than mice fed moderate (0.61-0.98 μM) or high (0.77-1.1 μM) Zn diets (SDs 0.1-0.4, n = 8-10). Smoke exposure increased plasma and BAL labile Zn (1.5-2.5 fold, P < 0.001), bronchoalveolar macrophages (2.0 fold, P < 0.0001) and MT-1 (1.5 fold), MIP-2 (2.3 fold) and MMP-12 (3.5 fold) mRNA. Zn supplementation reduced alveolar macrophage numbers by 62 and 52% in sham and smoke-exposed mice, respectively (Zn effect: P = 0.011). Gastrocnemius, soleus and tibialis anterior muscle mass were affected by both smoke and dietary Zn in the order of 3-7%. The 50-60% reduction in alveolar macrophages in Zn-supplemented mice supports our evolving hypothesis that Zn is an important anti-inflammatory mediator of airway inflammation. Restoring airway Zn levels through dietary supplementation may lessen the severity of lung inflammation when Zn intake is low.Carol J. Lang, Michelle Hansen, Eugene Roscioli, Jessica Jones, Chiara Murgia, Margaret Leigh Ackland, Peter Zalewski, Gary Anderson, Richard Ruffi

Disrupted epithelial/macrophage crosstalk via Spinster homologue 2-mediated S1P signaling may drive defective macrophage phagocytic function in COPD.

We have previously established a link between impaired phagocytic capacity and deregulated S1P signaling in alveolar macrophages from COPD subjects. We hypothesize that this defect may include a disruption of epithelial-macrophage crosstalk via Spns2-mediated intercellular S1P signaling.Primary alveolar macrophages and bronchial epithelial cells from COPD subjects and controls, cell lines, and a mouse model of chronic cigarette smoke exposure were studied. Cells were exposed to 10% cigarette smoke extract, or vehicle control. Spns2 expression and subcellular localization was studied by immunofluorescence, confocal microscopy and RT-PCR. Phagocytosis was assessed by flow-cytometry. Levels of intra- and extracellular S1P were measured by S1P [3H]-labeling.Spns2 expression was significantly increased (p<0.05) in alveolar macrophages from current-smokers/COPD patients (n = 5) compared to healthy nonsmokers (n = 8) and non-smoker lung transplant patients (n = 4). Consistent with this finding, cigarette smoke induced a significant increase in Spns2 expression in both human alveolar and THP-1 macrophages. In contrast, a remarkable Spns2 down-regulation was noted in response to cigarette smoke in 16HBE14o- cell line (p<0.001 in 3 experiments), primary nasal epithelial cells (p<0.01 in 2 experiments), and in smoke-exposed mice (p<0.001, n = 6 animals per group). Spns2 was localized to cilia in primary bronchial epithelial cells. In both macrophage and epithelial cell types, Spns2 was also found localized to cytoplasm and the nucleus, in line with a predicted bipartile Nuclear Localization Signal at the position aa282 of the human Spns2 sequence. In smoke-exposed mice, alveolar macrophage phagocytic function positively correlated with Spns2 protein expression in bronchial epithelial cells.Our data suggest that the epithelium may be the major source for extracellular S1P in the airway and that there is a possible disruption of epithelial/macrophage cross talk via Spns2-mediated S1P signaling in COPD and in response to cigarette smoke exposure

Apical Localization of Zinc Transporter ZnT4 in Human Airway Epithelial Cells and Its Loss in a Murine Model of Allergic Airway Inflammation

The apical cytoplasm of airway epithelium (AE) contains abundant labile zinc (Zn) ions that are involved in the protection of AE from oxidants and inhaled noxious substances. A major question is how dietary Zn traffics to this compartment. In rat airways, in vivo selenite autometallographic (Se-AMG)-electron microscopy revealed labile Zn-selenium nanocrystals in structures resembling secretory vesicles in the apical cytoplasm. This observation was consistent with the starry-sky Zinquin fluorescence staining of labile Zn ions confined to the same region. The vesicular Zn transporter ZnT4 was likewise prominent in both the apical and basal parts of the epithelium both in rodent and human AE, although the apical pools were more obvious. Expression of ZnT4 mRNA was unaffected by changes in the extracellular Zn concentration. However, levels increased 3-fold during growth of cells in air liquid interface cultures and decreased sharply in the presence of retinoic acid. When comparing nasal versus bronchial human AE cells, there were significant positive correlations between levels of ZnT4 from the same subject, suggesting that nasal brushings may allow monitoring of airway Zn transporter expression. Finally, there were marked losses of both basally-located ZnT4 protein and labile Zn in the bronchial epithelium of mice with allergic airway inflammation. This study is the first to describe co-localization of zinc vesicles with the specific zinc transporter ZnT4 in airway epithelium and loss of ZnT4 protein in inflamed airways. Direct evidence that ZnT4 regulates Zn levels in the epithelium still needs to be provided. We speculate that ZnT4 is an important regulator of zinc ion accumulation in secretory apical vesicles and that the loss of labile Zn and ZnT4 in airway inflammation contributes to AE vulnerability in diseases such as asthma.Other UBCNon UBCReviewedFacult

Relative mRNA expression levels of <i>S1PR2</i> and <i>S1PR5</i> in alveolar macrophages.

<p>(A) Significantly higher mRNA expression levels of <i>S1PR2</i> in current healthy smokers (9.3-fold increase), current smoker COPD subjects (6.1-fold increase) and in the total COPD group (4.3-fold increase) versus control subjects (*p<0.05 vs control) was found. No significant increase of <i>S1PR2</i> was found in ex-smoker COPD patients compared to control subjects.<b> (B)</b> Significantly higher mRNA expression of <i>S1PR5</i> was noted in in ex-smoker COPD subjects (32-fold increase), current smoker COPD subjects (5.4-fold increase) and in the COPD group (14.6-fold increase) versus healthy control subjects (*p<0.05 vs control). Data presented as box plots as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122771#pone.0122771.g001" target="_blank">Fig 1</a>. The Ct values were subtracted from 20 so that higher values mean higher mRNA expression levels.</p

Effects of antagonising <i>S1PR3</i> and <i>S1PR5</i> with Suramin.

<p>To further elucidate functional effect of increased <i>S1PR5</i> on macrophage phagocytic ability, we performed the phagocytosis assay in the presence of varying concentrations of Suramin, an antagonist of <i>S1PR3</i> and <i>S1PR5</i>. Suramin at concentrations of 10nM to 10μM were added for 30 min prior to the phagocytosis assay. We noted a significant (p<0.05) increase in phagocytosis of apoptotic cells in the presence of Suramin.</p

Patient demographics.

<p>Data are presented as number, or median and data range. COPD: chronic obstructive pulmonary disease; Smoking history: cur: current, ex: ex-smokers, no: never smoked; Lung Cancer: presence of lung cancer; NSCLC: Non-small cell lung cancer; SCLC: Small cell lung cancer; Type of NSCLC: adeno: adenocarcinoma, squa: squamous cell carcinoma, larg: large cell carcinoma; Radiation therapy: cur: current, pre: previous exposure, no: never exposed; Chemotherapy: cur: current, pre: previous exposure, no: never exposed; Pky: smoking pack years;fev₁: forced expiratory volume in one second, % pred: percentage of predicted; fvc: forced vital capacity.</p><p>*: significant difference from control group, p<0.05. Note that one COPD patient was classified as ‘mild COPD’, with a fev₁/fvc of 67.</p><p>Patient demographics.</p

Correlations between the relative mRNA expression of various components of the S1P signalling system and lung function (fev₁).

<p>Significant (p<0.05) correlations were found between fev₁ and <b>(A)</b><i>SPHK1</i><b>(B)</b><i>S1PR1</i><b>(C)</b><i>S1PR3</i>, <b>(D)</b><i>S1PR5</i>, p<0.05. The correlation coefficients are listed in the results.</p

Relative mRNA expression of the S1P synthesizing enzymes, <i>SPHK1</i>, and <i>SPHK2</i> in alveolar macrophages.

<p><b>(A)</b> Significantly higher mRNA expression of <i>SPHK1</i> was noted in current healthy smokers (6-fold increase), current smoker COPD (4.8-fold increase) and total COPD (3.5-fold increase) compared to control subjects (*p<0.05 vs control). No significant increase of <i>SPHK1</i> was found in alveolar macrophages isolated from ex-smoker COPD patients.<b> (B)</b> Significantly higher mRNA expression of <i>SPHK2</i> in current healthy smokers (4.4-fold increase), current smoker COPD(2.3-fold increase) and in the total COPD group (2.1-fold increase) versus control subjects (*p<0.05 vs control) was found. No significant increase in <i>SPHK2</i> was found in ex-smoker COPD subjects compared to control subjects. Data presented as box plots as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122771#pone.0122771.g001" target="_blank">Fig 1</a>. The Ct values were subtracted from 20 so that higher values mean higher mRNA expression levels. Data presented as box plots as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122771#pone.0122771.g001" target="_blank">Fig 1</a>. The Ct values were subtracted from 20 so that higher values represent higher mRNA expression levels.</p