Dual Exposure to E-Cigarette Vapour and Cigarette Smoke Results in Poorer Airway Cell, Monocyte, and Macrophage Function Than Single Exposure.

E-cigarette users predominantly also continue to smoke cigarettes. These Dual Users either consume e-cigarettes in locations where smoking is not allowed, but vaping is, or to reduce their consumption of cigarettes, believing it will lead to harm reduction. Whilst it is known that e-cigarette vapour is chemically less complex than cigarette smoke, it has a distinct chemical profile, and very little is known about the health impacts of exposure to both chemical profiles vs. either alone. We simultaneously exposed cells in vitro to non-toxic levels of e-cigarette vapour extract (EVE) and cigarette smoke extract (CSE) to determine their effects on 16HBE14o- airway epithelial cell metabolism and inflammatory response, as well as immune cell (THP-1 cells and monocyte-derived macrophages (MDM) from healthy volunteers) migration, phagocytosis, and inflammatory response. We observed increased toxicity, reduced metabolism (a marker of proliferation) in airway epithelial cells, and reduced monocyte migration, macrophage phagocytosis, and altered chemokine production after exposure to either CSE or EVE. These cellular responses were greater after dual exposure to CSE and EVE. The airway epithelial cells from smokers showed reduced metabolism after EVE (the Switcher model) and dual CSE and EVE exposure. When EVE and CSE were allowed to interact, the chemicals were found to be altered, and new chemicals were also found compared to the CSE and EVE profiles. Dual exposure to e-cigarette vapour and cigarette smoke led to worse functional outcomes in cells compared to either single exposure alone, adding to limited data that dual use may be more dangerous than smoking only.

Effects of E-cigarette E-liquid components on bronchial epithelial cells: Demonstration of dysfunctional efferocytosis.

BACKGROUND AND OBJECTIVE: E-cigarettes are often marketed and thought of as emitting harmless vapour; however, verification of their safety for non-smokers is scarce. We have previously shown that E-cigarettes cause decreased phagocytosis of bacteria by macrophages via reductions in surface bacterial recognition receptors. This study assessed the effect of E-cigarette constituents, 3 E-liquid apple flavours, nicotine, vegetable glycerine and propylene glycol, on bronchial epithelial cell viability, apoptosis and cytokine secretion and macrophage phagocytosis of apoptotic airway cells and phagocytic recognition molecules. METHODS: Cell necrosis and apoptosis were measured by Sytox Green stain and Annexin V. Efferocytosis was measured by internalization of pHrodo Green labelled apoptotic airway cells by macrophages. Expression of macrophage cell surface apoptotic cell receptors was measured by flow cytometry. Cytokine release by E-cigarette-exposed airway cells was measured by cytokine bead array. RESULTS: E-cigarette vapour increased primary bronchial epithelial necrosis and apoptosis. E-cigarette vapour reduced efferocytosis (lowest flavour 12.1%) versus control (20.2%, P = 0.032). The efferocytosis receptor CD44 was reduced by one flavour (MFI 1863 vs 2332 control, P = 0.016) and all components reduced expression of CD36, including the glycol bases (MFI 1067-12 274 vs 1415 control). Reduced secretion of TNF-α, IL-6, IP-10, MIP-1α and MIP-1ß was observed for all flavour variants. CONCLUSION: E-cigarettes can cause bronchial epithelial apoptosis and macrophage efferocytosis dysfunction via reduced expression of apoptotic cell recognition receptors. These data further show that E-cigarettes should not be considered harmless to non-smokers and their effects may go far beyond cytotoxicity to cells.

Airway epithelial cells exposed to wildfire smoke extract exhibit dysregulated autophagy and barrier dysfunction consistent with COPD.

BACKGROUND: Individuals with respiratory disease are being increasingly exposed to wildfire smoke as populations encroach further into forested regions and climate change continues to bring higher temperatures with lower rainfall. Frequent exposures have significant potential to accelerate conditions such as chronic obstructive pulmonary disease (COPD) which is characterised by an exaggerated inflammatory response to environmental stimuli. Here we employ models of human airway epithelium exposed to wildfire smoke-extract (WFSE) to examine modulation in airway epithelial cell (AEC) survival, fragility and barrier function. METHODS: Submerged cultures of small airway epithelial cells (SAEC) and differentiated air-liquid interface (ALI) cultures of primary bronchial AEC (bAEC) were treated for 1-24 h with 1-10% WFSE generated from plant species found in the Australian bushland. Autophagy (LC3-II and Sequestosome), apoptosis (Poly-(ADP)-Ribose Polymerase (PARP) cleavage) and tight junction proteins were measured using western blot. Barrier function was assessed via permeability of fluorescein tracers and measuring trans-epithelial electrical resistance. The production of IL-6 was assessed using ELISA. RESULTS: Primary epithelial models exposed to WFSE exhibited a significant blockade in autophagy as evidenced by an increase in LC3-II coupled with a concomitant elevation in Sequestosome abundance. These exposures also induced significant PARP cleavage indicative of apoptotic changes. ALI cultures of bAEC treated with 5% WFSE demonstrated barrier dysfunction with significant increases in paracellular molecular permeability and ionic conductance, and a reduction in the abundance of the tight junction proteins ZO-1 and Claudin-1. These cultures also exhibited increased IL-6 secretion consistent with the aberrant and pro-inflammatory repair response observed in the COPD airways. Further, blocks in autophagy and barrier disruption were significantly elevated in response to WFSE in comparison to similar exposures with cigarette smoke-extract. CONCLUSION: WFSE inhibits autophagic flux and induces barrier dysfunction in the airway epithelium. As autophagy is a central regulator of cellular repair, viability, and inflammation, targeting the block in autophagic flux may ameliorate the consequences of wildfire smoke-exposure for individuals with pre-existing respiratory conditions.

Nonantibiotic macrolides restore airway macrophage phagocytic function with potential anti-inflammatory effects in chronic lung diseases.

We reported defective efferocytosis associated with cigarette smoking and/or airway inflammation in chronic lung diseases, including chronic obstructive pulmonary disease, severe asthma, and childhood bronchiectasis. We also showed defects in phagocytosis of nontypeable Haemophilus influenzae (NTHi), a common colonizer of the lower airway in these diseases. These defects could be substantially overcome with low-dose azithromycin; however, chronic use may induce bacterial resistance. The aim of the present study was therefore to investigate two novel macrolides-2'-desoxy-9-(S)-erythromycylamine (GS-459755) and azithromycin-based 2'-desoxy molecule (GS-560660)-with significantly diminished antibiotic activity against Staphylococcus aureus, Streptococcus pneumonia, Moraxella catarrhalis, and H. influenzae We tested their effects on efferocytosis, phagocytosis of NTHi, cell viability, receptors involved in recognition of apoptotic cells and/or NTHi (flow cytometry), secreted and cleaved intracellular IL-1ß (cytometric bead array, immunofluorescence/confocal microscopy), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) using primary alveolar macrophages and THP-1 macrophages ± 10% cigarette smoke extract. Dose-response experiments showed optimal prophagocytic effects of GS-459755 and GS-560660 at concentrations of 0.5-1 µg/ml compared with our findings with azithromycin. Both macrolides significantly improved phagocytosis of apoptotic cells and NTHi (e.g., increases in efferocytosis and phagocytosis of NTHi: GS-459755, 23 and 22.5%, P = 0.043; GS-560660, 23.5 and 22%, P = 0.043, respectively). Macrophage viability remained >85% following 24 h exposure to either macrolide at concentrations up to 20 µg/ml. Secreted and intracellular-cleaved IL-1ß was decreased with both macrolides with no significant changes in recognition molecules c-mer proto-oncogene tyrosine kinase; scavenger receptor class A, member 1; Toll-like receptor 2/4; or CD36. Particulate cytoplasmic immunofluorescence of NLRP3 inflammasome was also reduced significantly. We conclude that GS-459755 and GS-560660 may be useful for reducing airway inflammation in chronic lung diseases without inducing bacterial resistance.

BIRC3 single nucleotide polymorphism associate with asthma susceptibility and the abundance of eosinophils and neutrophils.

BACKGROUND AND OBJECTIVE: Aberrant apoptosis is a disease susceptibility mechanism relevant for asthma, whereby fragility of the airway epithelium and enhanced survival of inflammatory cells, contributes to its pathogenesis and prolongation. Cellular Inhibitor of Apoptosis Proteins (cIAP) suppress apoptosis, and participate in the immune response. In this study, single nucleotide polymorphisms (SNP) in the BIRC2 (codes cIAP1) and BIRC3 (cIAP2) genes were evaluated for an association with asthma. METHODS: Caucasian asthmatic (n = 203) and control (n = 198) subjects were selected from participants in the North West Adelaide Health Study. SNPs (n = 9) spanning the consecutively positioned BIRC2 and BIRC3 genes, were selected using a haplotype tagging approach. Alleles and haplotype associations were analysed by logistic regression, assuming an additive genetic model, and adjusted for gender and atopy. RESULTS: The frequency of the minor allele for the BIRC3 SNP rs3460 was significantly lower in asthmatics compared to the control cases (P = 0.046). BIRC3 SNPs rs7928663 and rs7127583 associated with a reduction in eosinophil and neutrophil abundance when assessed across the study population (multivariate P values = 0.002, and 0.005, respectively). Further, the frequency of a haplotype tagged by rs3460, rs7928663 and rs7127583 was reduced in the asthma sub group (P = 0.05), while the presence of the major allele for rs7928663 associated with an increased load of circulating eosinophils and neutrophils (multivariate P value = 0.001). CONCLUSIONS: Polymorphisms in the BIRC3 gene, but not BIRC2, are associated with a protective effect with regards to asthma susceptibility, and a reduced load of inflammatory cells.

Zinc-rich inhibitor of apoptosis proteins (IAPs) as regulatory factors in the epithelium of normal and inflamed airways.

Integrity of the airway epithelium (AE) is important in the context of inhaled allergens and noxious substances, particularly during asthma-related airway inflammation where there is increased vulnerability of the AE to cell death. Apoptosis involves a number of signaling pathways which activate procaspases leading to cleavage of critical substrates. Understanding the factors which regulate AE caspases is important for development of strategies to minimize AE damage and airway inflammation, and therefore to better control asthma. One such factor is the essential dietary metal zinc. Zinc deficiency results in enhanced AE apoptosis, and worsened airway inflammation. This has implications for asthma, where abnormalities in zinc homeostasis have been observed. Zinc is thought to suppress the steps involved in caspase-3 activation. One target of zinc is the family of inhibitor of apoptosis proteins (IAPs) which are endogenous regulators of caspases. More studies are needed to identify the roles of IAPs in regulating apoptosis in normal and inflamed airways and to study their interaction with labile zinc ions. This new information will provide a framework for future clinical studies aimed at monitoring and management of airway zinc levels as well as minimising airway damage and inflammation in asthma.

X-linked inhibitor of apoptosis single nucleotide polymorphisms and copy number variation are not risk factors for asthma.

BACKGROUND AND OBJECTIVE: Aberrant apoptosis in asthma contributes to airway inflammation. Early apoptosis and fragility of airway epithelial cells and delayed apoptosis of inflammatory lymphocytes can cooperate to increase airway inflammation. In this study, single nucleotide polymorphisms (SNPs) and copy number variation (CNV) in the Baculoviral inhibitor of apoptosis protein repeat-containing 4 (BIRC4) gene (which encodes X-linked inhibitor of apoptosis protein) were evaluated for associations with asthma. METHODS: Asthma cases (n = 203) were identified from Caucasian cohort participants in the North West Adelaide Health Study and matched with 198 controls. Asthma status was defined using self-report of doctor-diagnosed asthma, in conjunction with spirometry and bronchodilator response. Seven SNPs, which spanned the entire BIRC4 gene, were selected for the study on the basis of a haplotype tagging approach. SNPs genotyping was performed on the SEQUENOM MassARRAY iPLEX Gold platform, and genotyping success rate was > 98%. BIRC4 gene CNV was measured using a duplex Taqman qPCR assay, with RNAseP as the reference gene. Alleles and haplotype associations were analysed by logistic regression, assuming an additive genetic model, and adjusted for gender and atopy. RESULTS: BIRC4 gene copy number was determined entirely by gender. All SNPs were in Hardy-Weinberg equilibrium for both case and control females. BIRC4 allele and haplotype frequencies were comparable between asthma cases and controls. CONCLUSIONS: There is no evidence of CNV in BIRC4, and BIRC4 is not a susceptibility gene for asthma.

E-Cigarette Vapour Increases ACE2 and TMPRSS2 Expression in a Flavour- and Nicotine-Dependent Manner.

COVID-19 infects via the respiratory system, but it can affect multiple systems and lead to multi system failure. There is growing evidence that smoking may be associated with higher rates of COVID-19 infections and worse outcomes due to increased levels of ACE2 in lung epithelial cells, but it is unknown whether E-cigarette use may lead to increased risk of COVID-19 infection from the SARS-CoV-2 virus. In this study, healthy donor bronchial epithelial cells (NHBE) and monocyte-derived macrophages (MDM) were exposed to cigarette smoke extract (CSE) or nicotine or flavoured E-cigarette vapour extract (EVE) before the assessment of SARS-CoV-2 recognition receptors ACE2 and TMPRSS2 genes. MDMs exposed to CSE and Tobacco EVE showed increased ACE2 expression; however, no treatment altered the TMPRSS2 expression. ACE2 was found to be upregulated by >2-fold in NHBE cells exposed to CSE, as well as nicotine, banana, or chocolate EVE, while TMPRSS2 was only upregulated by CSE or nicotine EVE exposure. These findings suggesting that flavourings can increase ACE2 expression in multiple cell types, while TMPRSS2 expression increases are limited to the epithelial cells in airways and may be limited to nicotine and/or cigarette smoke exposure. Therefore, increased risk of COVID-19 infection cannot be ruled out for vapers.

AIM2 nuclear exit and inflammasome activation in chronic obstructive pulmonary disease and response to cigarette smoke.

INTRODUCTION: The role inflammasomes play in chronic obstructive pulmonary disease (COPD) is unclear. We hypothesised that the AIM2 inflammasome is activated in the airways of COPD patients, and in response to cigarette smoke. METHODS: Lung tissue, bronchoscopy-derived alveolar macrophages and bronchial epithelial cells from COPD patients and healthy donors; lungs from cigarette smoke-exposed mice; and cigarette smoke extract-stimulated alveolar macrophages from healthy controls and HBEC30KT cell line were investigated. AIM2 inflammasome activation was assessed by multi-fluorescence quantitative confocal microscopy of speck foci positive for AIM2, inflammasome component ASC and cleaved IL-1ß. Subcellular AIM2 localization was assessed by confocal microscopy, and immunoblot of fractionated cell lysates. Nuclear localization was supported by in-silico analysis of nuclear localization predicted scores of peptide sequences. Nuclear and cytoplasmic AIM2 was demonstrated by immunoblot in both cellular fractions from HBEC30KT cells. RESULTS: Increased cytoplasmic AIM2 speck foci, colocalized with cleaved IL-1ß, were demonstrated in COPD lungs (n = 9) vs. control (n = 5), showing significant positive correlations with GOLD stages. AIM2 nuclear-to-cytoplasmic redistribution was demonstrated in bronchiolar epithelium in cigarette-exposed mice and in HBEC30KT cells post 24 h stimulation with 5% cigarette smoke extract. Alveolar macrophages from 8 healthy non-smokers responded to cigarette smoke extract with an > 8-fold increase (p < 0.05) of cytoplasmic AIM2 and > 6-fold increase (p < 0.01) of colocalized cleaved IL-1ß speck foci, which were also localized with ASC. CONCLUSION: The AIM2 inflammasome is activated in the airway of COPD patients, and in response to cigarette smoke exposure, associated with a nuclear to cytoplasmic shift in the distribution of AIM2.

Interventional low-dose azithromycin attenuates cigarette smoke-induced emphysema and lung inflammation in mice.

Cigarette smoke (CS)-induced emphysema is an important contributor to chronic obstructive pulmonary disease (COPD). We have shown the efficacy of azithromycin in reducing airway inflammation in COPD and in reducing exacerbations in severe asthma; however, the effects of long-term azithromycin on emphysema development have not been shown. We employed live animal imaging to monitor emphysema-like development and the effects of interventional azithromycin treatment in CS-exposed mice. BALB/c mice (female, 10 weeks; n = 10) were exposed to CS for 1 hr twice daily, 5 days/week, and for 12 weeks (CS). Half were cotreated with low-dose azithromycin during weeks 7-12 (CS + Azi; 0.2 mg kg-1  day-1 ). Microcomputed tomography (CT) and magnetic resonance imaging (MRI) scans were acquired longitudinally. Histological examinations were performed post mortem (mean linear intercept (Lm) and leukocyte infiltration). CS increased median Lm (CS: 42.45 µm versus control: 34.7 µm; p = .0317), this was recovered in CS + Azi mice (33.03 µm). Average CT values were reduced in CS mice (CS: -399.5 Hounsfield units (HU) versus control: -384.9 HU; p = .0286) but not in CS + Azi mice (-377.3 HU). CT values negatively correlated with Lm (r = -.7972; p = .0029) and T2 -weighted MRI (r = -.6434; p = .0278). MRI also showed significant CS-induced inflammatory changes that were attenuated by azithromycin in the lungs, and positively correlated with Lm (r = .7622; p = .0055) and inflammatory foci counts (r = .6503; p = .0257). Monitoring of emphysema development is possible via micro-CT and MRI. Interventional azithromycin treatment in CS-exposed mice attenuated the development of pulmonary emphysema-like changes.

Sphingosine signaling dysfunction in airway cells as a potential contributor to progression from protracted bacterial bronchitis to bronchiectasis in children.

AIM: Protracted bacterial bronchitis (PBB) is considered a potential precursor to bronchiectasis (BE) in some children. We previously showed that alveolar macrophages (AM) from children with PBB or BE have a similar significant defect in phagocytic capacity, with proinflammatory associations. We hypothesized that the mechanisms responsible for this defect involve dysregulation of the sphingosine-1-phosphate (S1P) signaling pathway, as we have found in adult inflammatory lung diseases. METHOD: We employed a Custom TaqMan OpenArray to investigate gene expression of S1P-generating enzymes: sphingosine kinases (SPHK) 1/2, S1P phosphatase 2 (SGPP2), S1P lyase 1 (SGPL1), S1P receptors (S1PR) 1/2/4/5; proinflammatory cytokines TNF-α (TNF) and IFNγ (IFNG), the cytotoxic mediator granzyme B (GZMB), and inflammasomes AIM2 and NLRP3, in bronchoalveolar lavage from 15 children with BE, 15 with PBB and 17 age-matched controls, and determined association with clinical/demographic variables and airway inflammation. RESULT: Significantly increased expression of S1PR1, S1PR2, and SPHK1 was noted in PBB and BE AM vs controls with increased SGPP2 only in PBB. TNF, IFNG, AIM2, and NLRP3 were significantly increased in both disease groups with increased GZMB only in PBB. There were no significant differences in the expression of any other S1P-related mediator between groups. There were significant positive associations between Haemophilus influenzae growth and expression of S1PR1 and NLRP3; between S1PR1 and S1PR2, NLRP3 and IFNG; between S1PR2 and AIM2, SPHK1, and SPHK2; and between SPHK1 and GZMB, IFNG, AIM2, and NLRP3. CONCLUSION: Children with PBB and BE share similar S1P-associated gene expression profiles. AM phagocytic dysfunction and inflammation in these children may occur due to dysregulated S1P signaling.

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-1ß, 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.

Phagocytosis and Inflammation: Exploring the effects of the components of E-cigarette vapor on macrophages.

E-cigarettes are perceived as harmless; however, evidence of their safety is lacking. New data suggests E-cigarettes discharge a range of compounds capable of physiological damage to users. We previously established that cigarette smoke caused defective alveolar macrophage phagocytosis. The present study compared the effect E-cigarette of components; E-liquid flavors, nicotine, vegetable glycerine, and propylene glycol on phagocytosis, proinflammatory cytokine secretion, and phagocytic recognition molecule expression using differentiated THP-1 macrophages. Similar to CSE, phagocytosis of NTHi bacteria was significantly decreased by E-liquid flavoring (11.65-15.75%) versus control (27.01%). Nicotine also decreased phagocytosis (15.26%). E-liquid, nicotine, and E-liquid+ nicotine reduced phagocytic recognition molecules; SR-A1 and TLR-2. IL-8 secretion increased with flavor and nicotine, while TNFα, IL-1ß, IL-6, MIP-1α, MIP-1ß, and MCP-1 decreased after exposure to most flavors and nicotine. PG, VG, or PG:VG mix also induced a decrease in MIP-1α and MIP-1ß We conclude that E-cigarettes can cause macrophage phagocytic dysfunction, expression of phagocytic recognition receptors and cytokine secretion pathways. As such, E-cigarettes should be treated with caution by users, especially those who are nonsmokers.

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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Cigarette smoke inhibits efferocytosis via deregulation of sphingosine kinase signaling: reversal with exogenous S1P and the S1P analogue FTY720.

Alveolar macrophages from chronic obstructive pulmonary disease patients and cigarette smokers are deficient in their ability to phagocytose apoptotic bronchial epithelial cells (efferocytosis). We hypothesized that the defect is mediated via inhibition of sphingosine kinases and/or their subcellular mislocalization in response to cigarette smoke and can be normalized with exogenous sphingosine-1-phosphate or FTY720 (fingolimod), a modulator of sphingosine-1-phosphate signaling, which has been shown to be clinically useful in multiple sclerosis. Measurement of sphingosine kinase 1/2 activities by [(32)P]-labeled sphingosine-1-phosphate revealed a 30% reduction of sphingosine kinase 1 (P < 0.05) and a nonsignificant decrease of sphingosine kinase 2 in THP-1 macrophages after 1 h cigarette smoke extract exposure. By confocal analysis macrophage sphingosine kinase 1 protein was normally localized to the plasma membrane and cytoplasm and sphingosine kinase 2 to the nucleus and cytoplasm but absent at the cell surface. Cigarette smoke extract exposure (24 h) led to a retraction of sphingosine kinase 1 from the plasma membrane and sphingosine kinase 1/2 clumping in the Golgi domain. Selective inhibition of sphingosine kinase 2 with 25 µM ABC294640 led to 36% inhibition of efferocytosis (P < 0.05); 10 µM sphingosine kinase inhibitor/5C (sphingosine kinase 1-selective inhibitor) induced a nonsignificant inhibition of efferocytosis, but its combination with ABC294640 led to 56% inhibition (P < 0.01 vs. control and < 0.05 vs. single inhibitors). Cigarette smoke-inhibited efferocytosis was significantly (P < 0.05) reversed to near-control levels in the presence of 10-100 nM exogenous sphingosine-1-phosphate or FTY720, and FTY720 reduced cigarette smoke-induced clumping of sphingosine kinase 1/2 in the Golgi domain. These data strongly support a role of sphingosine kinase 1/2 in efferocytosis and as novel therapeutic targets in chronic obstructive pulmonary disease.

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.

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.