Cellular inhibitor of apoptosis-2 is a critical regulator of apoptosis in airway epithelial cells treated with asthma-related inflammatory cytokines

Aberrant apoptosis of airway epithelial cells (AECs) is a disease contributing feature in the airways of asthmatics. The proinflammatory cytokines tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are increased in asthma and have been shown to contribute to apoptosis at the airways. In the present study, we investigated the role of the inhibitor of apoptosis protein (IAP) family in primary AECs exposed to TNFα and IFNγ. IAPs are potent regulators of caspase activity elicited by the intrinsic and extrinsic apoptosis pathways. However, while caspase‐mediated apoptosis was observed in AECs exposed to doxorubicin, it was not observed after cytokine treatment. Instead, AECs exhibited proapoptotic changes evidenced by an increased Bax:Bcl2 transcript ratio and partial processing of procaspase‐3. Examination by quantitative reverse transcription polymerase chain reaction (qRT‐PCR) and Western analysis showed that proapoptotic changes were associated with a time‐ and dose‐dependent induction of cellular IAP‐2 (cIAP2), potentiated primarily by IFNγ. The abundance of the IAP antagonists X‐linked IAP‐associated factor 1 (XAF1) and second mitochondria‐derived activator of caspases did not change, although a moderate nuclear redistribution was observed for XAF1, which was also observed for cIAP2. Small interfering RNA (siRNA)‐mediated depletion of cIAP2 from AECs leads to caspase‐3 activation and poly (ADP‐ribose) polymerase cleavage, but this required extended cytokine exposure to produce a concomitant decrease in cIAP1 and Bcl2. These results indicate that AECs possess endogenous mechanisms making them highly resistant to apoptosis due to asthma‐related inflammatory cytokines, and the activity of cIAP2 plays an important role in this protection.Eugene Roscioli, Rhys Hamon, Richard E. Ruffin, Susan Lester, and Peter Zalewsk

Bushfire smoke is pro-inflammatory and suppresses macrophage phagocytic function

Bushfires are increasing in frequency and severity worldwide. Bushfire smoke contains organic/inorganic compounds including aldehydes and acrolein. We described suppressive effects of tobacco smoke on the phagocytic capacity of airway macrophages, linked to secondary necrosis of uncleared apoptotic epithelial cells, persistence of non-typeable H. influenzae (NTHi), and inflammation. We hypothesised that bushfire smoke extract (BFSE) would similarly impair macrophage function. THP-1 or monocyte-derived macrophages (MDM) were exposed to 1-10% BFSE prepared from foliage of 5 common Australian native plants (genus Acacia or Eucalyptus), or 10% cigarette smoke extract (CSE). Phagocytic recognition receptors were measured by flow cytometry; pro-inflammatory cytokines and caspase 1 by immunofluorescence or cytometric bead array; viability by LDH assay; and capsase-3/PARP by western blot. BFSE significantly decreased phagocytosis of apoptotic cells or NTHi by both THP-1 macrophages and MDM vs air control, consistent with the effects of CSE. BFSE significantly decreased MDM expression of CD36, CD44, SR-A1, CD206 and TLR-2 and increased active IL-1β, caspase-1 and secreted IL-8. BFSE dose-dependently decreased THP-1 macrophage viability (5-fold increase in LDH at 10%) and significantly increased active caspase-3. BFSE impairs macrophage function to a similar extent as CSE, highlighting the need for further research, especially in patients with pre-existing lung disease.Rhys Hamon, Hai B. Tran, Eugene Roscioli, Miranda Ween, Hubertus Jersmann, Sandra Hodg

Zinc and zinc transporters in macrophages and their roles in efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis

Subcellular Euclidean distance measurements with multicolor fluorescence localization imaging in cultured cells

This protocol measures the 3D Euclidean distance (Δ3D) between two/three fluorescently labeled kinetochore components in fixed samples using Kinetochore Delta software (KiDv1.0.1, MATLAB based). Overestimation of mean Δ3D is corrected through a Bayesian algorithm, with ΔEC distances reflecting the ensemble average positions of fluorophores within a kinetochore population. This package also enables kinetochore categorization, which can be used to sub-sample kinetochores and measure ΔEC. Together, this allows the dynamic architecture of human kinetochores to be investigated (tested in hTERT-RPE1 cells)

Bayesian inference of multi-point macromolecular architecture mixtures at nanometre resolution

Gaussian spot fitting methods have significantly extended the spatial range where fluorescent microscopy can be used, with recent techniques approaching nanometre (nm) resolutions. However, small inter-fluorophore distances are systematically over-estimated for typical molecular scales. This bias can be corrected computationally, but current algorithms are limited to correcting distances between pairs of fluorophores. Here we present a flexible Bayesian computational approach that infers the distances and angles between multiple fluorophores and has several advantages over these previous methods. Specifically it improves confidence intervals for small lengths, estimates measurement errors of each fluorophore individually and infers the correlations between polygon lengths. The latter is essential for determining the full multi-fluorophore 3D architecture. We further developed the algorithm to infer the mixture composition of a heterogeneous population of multiple polygon states. We use our algorithm to analyse the 3D architecture of the human kinetochore, a macro-molecular complex that is essential for high fidelity chromosome segregation during cell division. Using triple fluorophore image data we unravel the mixture of kinetochore states during human mitosis, inferring the conformation of microtubule attached and unattached kinetochores and their proportions across mitosis. We demonstrate that the attachment conformation correlates with intersister tension and sister alignment to the metaphase plate

Lymphocyte senescence in COPD is associated with decreased histone deacetylase 2 expression by pro-inflammatory lymphocytes

Histone acetyltransferases (HAT) and histone deacetylases (HDAC) are enzymes that upregulate and down-regulate pro-inflammatory gene transcription respectively. HDAC2 is required by corticosteroids to switch off activated inflammatory genes and is reduced in lung macrophages in COPD. We have shown that COPD patients have increased steroid resistant CD28null (senescent) pro-inflammatory T and NKT-like peripheral blood cells (particularly CD8+ subsets) and we hypothesized that these changes would be associated with a loss of HDAC2 from these senescent pro-inflammatory lymphocytes.Blood was collected from 10 COPD and 10 aged-matched controls. Intracellular pro-inflammatory cytokines, IFNγ and TNFα, and expression of CD28, HDAC2 and HAT, were determined in lymphocyte subsets in the presence of ± 5 mg/ml theophylline (HDAC2 activator), 10 μM prednisolone and 2.5 ng/ml cyclosporine A (immunosuppressant), using flow cytometry.There was a loss of HDAC2 from CD28null CD8+ T and NKT-like cells in COPD. There was a significant negative correlation between HDAC2 expression and the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -.763, p < 0.001 for T-cell IFNγ). There was a synergistic upregulation of HDAC2 and associated decrease in pro-inflammatory cytokine production in CD28nullCD8+ T and NKT-like cells in the presence of 5 mg/L theophylline + 10(-6) M prednisolone or 2.5 ng/mL cyclosporine A (CsA).Lymphocyte senescence in COPD is associated with loss of HDAC2 in CD28nullCD8+ T and NKT-like cells. Alternative treatment options such as combined theophylline with low-dose CsA, that inhibit these pro-inflammatory cells, may reduce systemic inflammation in COPD.Greg Hodge, Hubertus Jersmann, Hai B. Tran, Eugene Roscioli, Mark Holmes, Paul N. Reynolds and Sandra Hodg

Zinc deficiency as a codeterminant for airway epithelial barrier dysfunction in an ex vivo model of COPD

There is now convincing evidence that the airway epithelium drives the pathogenesis of COPD. A major aspect of this is the disease-related reduction in barrier function that is potentiated by dysregulation of tight junction (TJ) protein complexes. However, a significant number of studies using in vitro smoke exposure models have not observed alterations in barrier permeability. We have previously shown that zinc (Zn) is an influential cytoprotective factor for the airway epithelium, and its depletion by cigarette smoke produces disease-related modifications consistent with inflammatory changes in COPD. We hypothesized that Zn deficiency is a significant co-stimulus with cigarette smoke extract (CSE) for potentiating the leaky barrier phenotype exhibited in COPD. We employed an ex vivo model of differentiated human airway epithelium exposed to Zn depletion and CSE to determine the contribution of Zn in maintaining normal epithelial permeability. Western blot analysis demonstrated a significant downregulation of the TJ proteins such as ZO-1 (-1.93-fold, P<0.05) and Claudin-1 (-3.37-fold, P<0.01) with the combination exposure. Assessment of barrier function via paracellular ionic conductance and tracer permeability also showed that Zn depletion was an important factor, which potentiated an increase in epithelial permeability (P<0.001 for both) compared to Zn depletion or CSE exposures in isolation. Visual inspection of the epithelium using transmission electron microscopy revealed a marked reduction in junction complexes between the adjacent airway epithelial cells treated with a combination of Zn depletion and CSE. These observations identify Zn deficiency as a significant codeterminant with CSE as a factor leading to an increase in airway epithelial permeability. Hence, as Zn dyshomeostasis has been reported in the airway epithelium exposed to chronic cigarette smoke and inflammation, targeting these phenomena may represent a promising strategy to ameliorate the leaky barrier phenotype that is synonymous with COPD.Eugene Roscioli, Hubertus PA Jersmann, Susan Lester, Arash Badiei, Andrew Fon, Peter Zalewski, Sandra Hodg

Organotypic sinonasal airway culture systems are predictive of the mucociliary phenotype produced by bronchial airway epithelial cells

Published online: 10 November 2022Differentiated air-liquid interface models are the current standard to assess the mucociliary phenotype using clinically-derived samples in a controlled environment. However, obtaining basal progenitor airway epithelial cells (AEC) from the lungs is invasive and resource-intensive. Hence, we applied a tissue engineering approach to generate organotypic sinonasal AEC (nAEC) epithelia to determine whether they are predictive of bronchial AEC (bAEC) models. Basal progenitor AEC were isolated from healthy participants using a cytological brushing method and differentiated into epithelia on transwells until the mucociliary phenotype was observed. Tissue architecture was assessed using H&E and alcian blue/Verhoeff-Van Gieson staining, immunofluorescence (for cilia via acetylated α-tubulin labelling) and scanning electron microscopy. Differentiation and the formation of tight-junctions were monitored over the culture period (day 1-32) by quantifying trans-epithelial electrical resistance. End point (day 32) tight junction protein expression was assessed using Western blot analysis of ZO-1, Occludin-1 and Claudin-1. Reverse transcription qPCR-array was used to assess immunomodulatory and autophagy-specific transcript profiles. All outcome measures were assessed using R-statistical software. Mucociliary architecture was comparable for nAEC and bAEC-derived cultures, e.g. cell density P = 0.55, epithelial height P = 0.88 and cilia abundance P = 0.41. Trans-epithelial electrical resistance measures were distinct from day 1-14, converged over days 16-32, and were statistically similar over the entire culture period (global P  0.05). Transcript analysis for inflammatory markers demonstrated significant variation between nAEC and bAEC epithelial cultures, and favoured increased abundance in the nAEC model (e.g. TGFβ and IL-1β; P < 0.05). Conversely, the abundance of autophagy-related transcripts were comparable and the range of outcome measures for either model exhibited a considerably more confined uncertainty distribution than those observed for the inflammatory markers. Organotypic air-liquid interface models of nAEC are predictive of outcomes related to barrier function, mucociliary architecture and autophagy gene activity in corresponding bAEC models. However, inflammatory markers exhibited wide variation which may be explained by the sentinel immunological surveillance role of the sinonasal epithelium.Juliette Delhove, Moayed Alawami, Matthew Macowan, Susan E. Lester, Phan T. Nguyen, Hubertus P. A. Jersmann, Paul N. Reynolds and Eugene Rosciol