key: cord-0260265-6ni66nn6
authors: Bell, Rachael L.; McAuley, Danny F.; Shyamsundar, Murali; O’Kane, Cecila M.; Dombrowski, Yvonne
title: E-cigarette vapour from base components propylene glycol and vegetable glycerine inhibits the inflammatory response in macrophages and epithelial cells
date: 2022-03-14
journal: bioRxiv
DOI: 10.1101/2022.03.11.483808
sha: 391d76ab0e3ba621f7bce3e1df7dc9385ffd4bd4
doc_id: 260265
cord_uid: 6ni66nn6

E-cigarettes are a highly popular nicotine replacement therapy in the process of smoking cessation. Despite this, research on the effects of E-vapours to human health remains limited. The popularity of vaping and mass production of cheap E-liquids has led to compromised safety regulations, with contaminants such as heavy metals and alkaloids detected in multiple liquids. Vaporised E-liquids increase cellular ROS generation and inflammatory cytokine release from pulmonary macrophages. This suggests that E-cigarette usage might activate inflammasomes. Common food additives vegetable glycerine (VG) and propylene glycol (PG) form the base of all E-liquids, but little is known about their inflammatory potential once inhaled. Here, the effect of base components PG and VG on inflammasome activation and cytokine release was investigated in macrophages and epithelial cells exposed to E-liquids and vaporised E-liquid extract (E-vapour). Base E-liquid and E-vapour did not induce cellular cytotoxicity and non-vapourised E-liquid had no effect on IL-8 release. However, basic PG/VG E-vapour inhibited both IL-8 release and conventional inflammasome activation by known inflammatory activators in macrophages and epithelial cells. These results propose a novel inhibitory effect of basic E-vapour components to inflammatory challenges.

Introduction vapour increased the expression of ACE2 protein in airway epithelia, which has been 66 linked to the development of COVID-19 (McAlinden., 2021). Nicotine itself increases 67 ACE2 expression (Maggie., 2021) and indeed cigarette smoke has also emerged as 68 a risk factor for COVID-19 development due to increased ACE2 expression. 69 Furthermore, a study which collected peripheral blood mononuclear cells from 70 'healthy' E-cigarette users found increased levels of key proteases linked to ACE2 71 expression and SARS-COV-2 infection (Kelesidis., 2022) . 72 In vitro studies have shown that E-liquids and E-vapours are not as benign as originally 73 believed. E-liquids and -vapours induce cellular toxicity (Farsalinos., 2013 & 2014, 74 Willershausen., 2014, Scheffler., 2015, Hwang., 2016, Leig., 2016, Rowell., 2017, 75 Ween., 2019), inflammatory cytokine release (Wu., 2014 , Cervellati., 2014 Rubenstein., 2015, Lerner., 2015 , Higham., 2016 , Leigh., 2016 , Khachatoorian., 2021 77 and impaired cellular function (Schweitzer., 2015 , Hwang., 2016 , Scott., 2018 Gómez., 2020) of airway epithelia and immune cells. Human alveolar macrophages 79 secreted increased pro-inflammatory cytokines IL-8, IL-6, and TNFα along with 80 reactive oxygen species (ROS) and were more susceptible to cell death when exposed 81 to E-cigarette vapour extracts (Scott., 2018) . The cell culture medium was then changed to 2.5% FBS on the day of the experiment.

To induce IL-8 release, cells were treated with TNFα at 10 ng/ml (Peprotech, London, 144 UK) for 24 hours. although not significantly, when A549 epithelial cells were exposed to TNFα in E-283 vapour extracts compared to TNFα in control media ( Figure 3A ).

To investigate if the reduction in IL-8 release was due to TNFα being blocked by E- The E-vapour mediated reduction in IL-8 release was not due to E-vapour blocking IL- 365 Next, the effects of basic E-vapour on priming and activation of inflammasomes was 366 investigated using primary macrophages from healthy donors. Macrophages were 367 exposed to basic E-vapour as described in the experimental design either in 368 conjunction with LPS ( Figure 6A ) or in conjunction with nigericin ( Figure 6B ).

There was an observed, but not significant, decrease in the amount of IL-1β released 370 from macrophages when E-vapour was present during either the inflammasome 371 priming or activation stage compared to when macrophages were primed and 372 activated with LPS and nigericin on their own ( Figure 6C ).

Additionally, active caspase-1, which requires inflammasome activity, was absent from 394 We found that basic E-vapour consistent of only PG and VG had an inhibitory effect 395 on activated cells (Figures 2-6) . These effects where not seen with un-vapourised E-396 liquids (Figure 2-3) . When E-liquid is vaporised, it is possible that emissions may be 

Neither basic E-vapour containing only PG and VG, or nicotine-containing E-vapour 435 or E-liquid were cytotoxic to epithelial cells or macrophages tested here (Figure 1) . 436 However, all E-vapours reduced IL-8 release from epithelial cells and macrophages, 437 both in naïve state and activated with TNFα (Figure 2 and 3) . Cigarette smoke also 438 reduced IL-8 release from primary macrophages and led to increased macrophage 439 death, which was not observed with E-vapour ( Figure 2) . Furthermore, we observed a 440 change in macrophage morphology exposed to nicotine-containing E-vapours processing in THP1 cells and C57BL/6 mice exposed to cigarettes smoke, and the 488 NLRP3 inflammasome was supressed in reaction to Candida albicans in a rat model 1). It is also important to note the structural differences between smoke and E-vapours. 

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