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

Dataset for: Phagocytosis and Inflammation: Exploring the effects of the components of E-cigarette vapour 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 flavours, nicotine, vegetable glycerine, and propylene glycol on phagocytosis, pro-inflammatory 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 flavouring (11.65-15.75%) vs. 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 flavour and nicotine, while TNFα, IL-1β, IL-6, MIP-1α, MIP-1β, and MCP-1 decreased after exposure to most flavours and nicotine. PG, VG, or PG:VG mix also induced a decrease in MIP-1α and MIP-1β. 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 non-smokers

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

Human bronchial epithelial cells abundantly express SPHK1, SPHK2 and Spns2.

<p><b>A,</b> SPHK1 (red). <b>B</b>, SPHK2 (red). <b>C</b>, Spns2 (red, LifeSpan BioSciences rabbit polyclonal antibody). Green in <b>A-C</b> was beta-actin. <b>D, E</b>, Spns2 (green, Santa Cruz goat polyclonal antibody) was expressed in cilia, and colocalized with SPHK1 (red, yellow being merged color of red and green) in the cytoplasm. <b>F</b>, a negative staining control incubated with conjugated antibodies alone. Blue in <b>A-F</b> was DAPI. Arrows in <b>C, D, E</b> indicate Spns2 expression in cilia. Scale bars are in micrometers. Images are representative of bronchial epithelial cells obtained from 3 different non-smoking donors.</p

Spns2 expression in human macrophages.

<p><b>A</b>, Expression and subcellular localization of Spns2 in alveolar macrophages. Spns2 (rabbit antibody) was labeled in red, beta-actin in green, blue was staining of nuclei. Spns2 was localized to plasma membrane (arrowhead), nucleus (arrow), and cytoplasm. <b>B</b>, Representative confocal images of Spns2 (goat antibody, green) in COPD vs. non-COPD alveolar macrophages, captured for quantitative analysis. For the highly intense Spns2 immunofluorescence in the given COPD sample not become saturated, the laser settings had to be set as low as the immunofluorescence in the non-COPD sample was nearly invisible for naked eyes. Scale bars in <b>A</b> and <b>B</b> are in micrometers. <b>C</b>, Significant increase of Spns2 immunofluorescence in alveolar macrophages of smokers/COPD patients (n = 5) compared to healthy control (C, n = 8, p<0.01) and non-COPD non-smoker transplant patients (Tx, n = 4, p<0.01). <b>D</b>, Cigarette smoke extract induced significant increase (**, p<0.01) of Spns2 immunofluorescence in primary alveolar macrophages obtained from non-smokers (n = 6). <b>E</b>, Cigarette smoke extract induced significant increase (***, p<0.001, 5 experiments) of Spns2 immunofluorescence in THP-1 macrophages. <b>F</b>, Cigarette smoke extract induced significant increase (*, p<0.05, 3 experiments) of Spns2 mRNAs in THP-1 macrophages.</p

Defective phagocytosis in lung macrophages of cigarette smoked mice was associated with a down-regulated expression of Spns2 in bronchial epithelia.

<p><b>A,</b> Representative confocal images of Spns2 in lung tissue of control and cigarette smoke (CS)-exposed mice, and a negative control staining (NEG). Spns2 was stained in red, nuclei in blue. Scale bars are in micrometers. <b>B,</b> Significant decrease of Spns2 (MFI, mean fluorescence intensity) in bronchial epithelia in CS-exposed vs. control mice (p<0.001, pooled data from 6 animals per group). <b>C,</b> Cigarette smoke exposure induced a significant decrease of efferocytosis (p<0.05, n = 6 per group) in lung macrophages of CS-exposed vs. control mice, and a nearly significant trend of reduced NTHi phagocytosis (p = 0.063). <b>D,</b> Correlation between NTHi phagocytic activity of pulmonary macrophages in individual mice and expression of Spns2 in their bronchial epithelia (pooled data from 12 mice, r = 0.685; p = 0.014 by Spearman’s rho test).</p

Zinc and Zinc Transporters in Macrophages and Their Roles in Efferocytosis in COPD

<div><p>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 <i>in vitro</i> 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 <i>ZIP1</i> mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter <i>ZIP2,</i> 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.</p></div

Zn transporter mRNA expression in THP-1 monocytes and macrophages in response to intracellular Zn depletion.

<p>THP-1 monocytes and PMA differentiated THP-1 macrophages were treated with 16 µM TPEN for 4 h. RNA was isolated and target genes were detected using Taqman probes, with HPRT-1 and 18S RNA used as endogenous controls for 3 separate experiments (n = 3). Data is presented as mean fold change (±95% CI) compared to control (untreated) cells. * p: <0.05, ** p: <0.01, *** p: <0.001.</p