key: cord-0310097-xee5yr35
authors: Sharma, Aditi; Lee, Jasper; Fonseca, Ayden G.; Moshensky, Alex; Kothari, Taha; Sayed, Ibrahim M.; Ibeawuchi, Stella-Rita; Pranadinata, Rama F.; Ear, Jason; Sahoo, Debashis; Crotty-Alexander, Laura E.; Ghosh, Pradipta; Das, Soumita
title: E-cigarettes compromise the gut barrier and trigger gut inflammation
date: 2020-07-30
journal: bioRxiv
DOI: 10.1101/2020.07.29.227348
sha: ef77d0eeacdf7727426ae71351b7248ece753224
doc_id: 310097
cord_uid: xee5yr35

E-cigarette and vaping device use continue to rise, particularly in adolescents and young adults, but the safety of inhaling the multitude of chemicals within e-cigarette aerosols has been questioned. While several studies have evaluated vaping effects on the lungs and heart; effects on the gastrointestinal tract remain unknown. Using established murine models of acute (1 week) and chronic (3 month) daily e-cigarette aerosol inhalation, both with nicotine-containing and vehicle control e-liquids, murine colon transcriptomics and organoid co-culture models, we assessed the effects of e-cigarette use on the gut barrier and mucosal health. Histologic analyses revealed that chronic exposure to nicotine-free e-cigarette aerosols induced mucosal inflammation. Transcriptome analyses revealed that chronic, but not acute, nicotine-free e-cigarette use significantly reduced expression of tight junction markers, including occluding, and drove expression of pro-inflammatory cytokines. Exposure of murine and human enteroid-derived monolayers (EDMs) to nicotine-free e-cigarette aerosols alone, or in co-culture with invasive E. coli, confirmed that repetitive exposure was sufficient to recapitulate the key findings observed in vivo, i.e., barrier-disruption, downregulation of occludin, inflammation, and an accentuated risk of and response to bacterial infection. These data highlight an unexpected harmful effect of e-cigarette use on the gut barrier and pinpoint non-nicotine chemical components common across >90% of e-cigarette e-liquids as the source of harm. Given the ever-expanding importance of the integrity of the gut barrier for host fitness, and impact of gut mucosal inflammation on a multitude of chronic diseases, these findings are broadly relevant to medicine and public health. SIGNIFICANCE The safety of electronic cigarettes has been questioned amidst emerging evidence that they may derail our immune system and increase our susceptibility to infections. Despite these insights, their impact on the most critical entity that separates trillions of microbes from the largest immune system in our body, i.e., the gut barrier, remains unexplored. Using a combination of mouse models, gut transcriptomics, and murine and human gut-derived organoids, here we show that chronic exposure to aerosols of electronic-cigarette disrupts the gut barrier, increases its susceptibility to bacterial infections and triggers inflammation. Given the importance of the gut barrier in the maintenance of immune homeostasis, these findings provide valuable insights into the potential long-term harmful effects of electronic cigarettes on health.

Electronic nicotine delivery systems (ENDS), commonly referred to as e-cigarettes and vaping devices, were introduced to the international market in 2007 (1). Since then, e-cigarettes have become widely popular in the United States (2) , primarily among the nation's youth. Rapid growth in consumption is fueled in large part due to successful social media-based marketing and availability of appealing flavors and sleek devices. Although a large focus of research and regulation has been on the nicotine delivery function of these devices, because of the high addictiveness and known adverse health effects of nicotine, the majority of e-liquids contain propylene glycol (PG), glycerol (Gly), flavorants and contaminants, all of which may cause their own adverse effects on health (3) (4) (5) (6) (7) (8) . Regardless of chemicals contained, the popularity of e-cigarettes is driven, in part, by the notion that e-cigarettes represent a riskfree alternative to combustible cigarettes, and hence, few regulations exist to control the quality and composition of the ingredients used in e-cigarettes (7, 9) .

This concept of risk-free use has recently been challenged (10) . For instance, morbidity and mortality among teenagers and young adults due to the e-cigarette or vaping product use-associated lung injury (EVALI) epidemic (11) created a public health crisis in 2019-2020. This disease was found to be primarily associated with a single (non-nicotine) chemical included in vaping liquids: vitamin E acetate (12) . Prior to the identification of EVALI, in vitro and ex vivo research studies had showed that e-liquids induce inflammatory responses and alter innate immune defenses in myeloid and primary airway epithelial cells (13) (14) (15) . In vivo studies in which C57BL/6 mice were exposed to e-cigarette aerosols for 2 weeks found impairment of both pulmonary bacterial and viral clearance (16, 17) , which raises alarm for increased susceptibility to influenza and coronavirus infections in particular. Evidence of pulmonary and systemic inflammation has also been found in the plasma (18) and bronchoalveolar lavage (19) samples from human e-cigarette-users, with elevated biomarkers of inflammation, e.g., (interleukin (IL)-1β, IL-6, IL-8, IL-13 and interferon (IFN)-γ. Furthermore, mechanisms that may connect the use of ecigarettes and an increased risk for cancer development, as well as their stimulatory effect on cancer progression, have been proposed (20) and e-cigarettes have been shown to induce DNA damage [independent of nicotine (8) ] while reducing repair pathways (21) . Tang et al. determined that chronic ecigarette use for one year led to increased development of lung cancer in mice (22) . Recently, a NIHfunded panel reviewed the current evidence and came to the consensus that all chemical components of e-cigarette and vaping aerosols have the potential to cause distinct health effects (23) , both similar to and disparate from those of nicotine or conventional tobacco smoke, both in the heart and lungs and throughout the body (23) .

Here we set out to assess the impact of e-cigarette aerosol inhalation (with or without nicotine) on the gastrointestinal (GI) tract. The gut is resident to diverse microbiota (symbionts and pathobionts) and its handling of and response to the same is known to regulate several chronic diseases such as inflammatory bowel diseases (IBD), obesity, cardiovascular diseases, cancers and rheumatoid arthritis (24) . Beyond the fact that e-cigarette use significantly modulates the oral microbiome by increasing the abundance of oral pathobionts (25) , but is not known to significantly impact the gut microbiome (26) , nothing is known as to how e-cigarette use may influence the gut barrier. Using a combination of mouse models, transcriptomics, and murine and human gut-derived organoids as ex vivo near-physiologic model systems, here we expose the hitherto unknown effects of e-cigarettes on the gastrointestinal tract and provide insights into the potential long-term effects of e-cigarettes on health.

Daily e-cigarette aerosol inhalation drives inflammation in the colon and reduces expression of genes related to barrier function. E-cigarettes function by heating liquid solutions and then pulling them through an atomizer to create an aerosol that is inhaled. To establish the in vivo effect of inhalation of these aerosols on the colon, the distal colon was harvested from mice exposed daily to e-cigarette aerosols at two time-points: 1 week (resembling acute exposure) and 3 months (resembling chronic exposure) (Fig 1A) . Because the most common chemicals in e-cigarette aerosols are nicotine and humectants (propylene glycol and glycerol), we utilized nicotine-free and nicotine-containing (6 mg/ml) e-liquids with a 70:30 ratio of propylene glycol and glycerol within a Kanger Subtank attached to a box Mod e-device, and used exposure chambers with room air for controls (Fig 1A) . H&E staining of distal colons from mice acutely exposed to nicotine-free (vehicle only) e-cigarette aerosols (e-cig) demonstrated small, infrequent patches of leukocyte infiltration in the submucosal layers (Fig 1B, left; asterisk). Acute exposure to nicotine containing aerosols (e-cig + nicotine) was associated with infrequent patches of epithelial erosions. By contrast, chronic e-cig exposure led to large submucosal inflammatory infiltrates within the colon (Fig 1B, right) . No inflammatory infiltrates were seen in air controls, while smaller and infrequent infiltrates were present in colons of mice exposed to e-cig + nicotine.

Markers of gut epithelial tight junctions, e.g., occludin (OCLN), zonula occludens (ZO)-1 (TJP1) and Claudin-2 (CLDN2) had significantly reduced gene expression in mice chronically exposed to nicotine-free aerosols (e-cig) compared to air controls (Fig 1C) . The fact that e-cig exposure affects the levels of Claudin-2, a major regulator of TJ-specific obliteration of the intercellular space (27) , but not its counterpart Claudin-1 (Fig 1C) , which is specialized for TJ integrity in the skin epidermis indicates that the effects of e-cig on TJs may be gut specific. No reduction was observed in any of the barrier function genes in mice exposed to nicotine-containing e-cigarettes. No significant differences were observed in levels of pro-inflammatory cytokines MCP1 or IL-8 in the chronically exposed mice ( Fig   S1) . Finally, no statistically significant differences were observed in the transcript levels of TJ markers and pro-inflammatory cytokines in acute exposures between any conditions (Fig S1) .

These findings indicate that chronic, but not acute exposure to aerosols of nicotine-free ecigarettes is sufficient to trigger inflammation in the gut and that such inflammation is associated with reduced expression of markers of epithelial TJs. Findings also suggest that concomitant exposure to nicotine may alleviate both phenotypes.

The integrity of the gut barrier is maintained by a complex cross-talk between the gut epithelial barrier and immune cells in the lamina propria. To determine if the observed decrease in colon TJ markers in mice exposed to e-cigs is a direct consequence of aerosols on the gut epithelial barrier, we used an ex vivo near-physiologic model system called the "gut-in-a-dish" (Fig 1D) . In this model, crypt-derived stem cells isolated from mouse colon (see the Materials and Methods section) were used to generate organoids and later differentiated into polarized enteriod-derived monolayers (EDMs). These EDMs have been validated as model systems that closely resemble the physiologic gut lining in which all cell types (enterocytes, goblet, paneth enteroendocrine, and tuft cells) are proportionately represented (28) (29) (30) (31) ). We exposed the basolateral surfaces of the murine colonic EDMs to e-cigarette aerosol-infused media (or air-infused media; as controls) and analyzed the integrity of the gut barrier using two readouts ( Fig 1D) : (i) paracellular permeability, as reflected by low trans-epithelial electrical resistance (TEER) and (ii) molecular characterization of epithelial TJs by looking at the localization of occludin; this integral membrane protein allows us to not just visualize but also quantify the degree of TJ disruption.

Exposure to nicotine-free e-cigarette aerosol media caused a significant drop in TEER (~% change value of -97.3±0.3%) compared to untreated (UN; -0.5±8.1%) and air-treated (1.4±9.7%) controls (Fig 1E) .

Findings indicate a significant increase in paracellular permeability upon exposure to e-cig when compared to untreated (p = 0.0002) and air-treated (p = 0.0004) controls (Fig 1E) . In e-cig exposed EDMs, confocal microscopy showed significantly increased "burst" tricellular TJs [these are specialized regions of the TJ where three or more cells come in contact (32) , are also the regions where TJdisruption can be visualized/assessed first (33)] by ~60.1±8.1% when compared to untreated (p = 0.0010) and air-treated (p = 0.0015) controls (Fig 1F-G) . These findings show that chemicals contained within the most basic e-cigarette aerosols disrupt the epithelial barrier. Because these effects were seen in in vitro assays on isolated gut epithelial monolayers in the absence of immune cells, findings also suggest a direct cause and effect relationship between e-cigarette aerosol components and epithelial barrier disruption. Because the chemicals used to make the e-liquids and e-cig aerosols used in these studies (propylene glycol and glycerol) are found in >99% of all e-cigarettes, these data broadly apply to e-cigarettes and vaping devices.

To determine the global impact of e-cigarettes on the gut, we next carried out RNA sequencing on the distal colons. While acute exposure to nicotine-free e-cigarettes did not significantly change gene expression in the colon (Fig S2A) , chronic exposure was associated with significant changes (Fig 2A) .

A differential expression analysis showed that chronic exposure to nicotine-free e-cigarettes was associated with a significant upregulation of 120 genes and downregulation of 75 genes (with a 30% false discovery rate, FDR) (Fig 2B; Table S1 ). Barring a handful of genes (arrowheads, Fig 2B, S2B) these differences were virtually reversed when mice were exposed to nicotine-containing e-cigarettes ( Fig 2B) . TJ markers occludin and ZO1 were downregulated by nicotine-free, but not nicotinecontaining e-cigarettes (Fig 2C-D) . Multiple pro-inflammatory cytokines were either elevated significantly (MCP1, IL-8, TNFα) or showed an increasing trend but did not reach significance (Cxcl2) (Fig 2E-H) . These RNA Seq findings are in agreement with our prior observations by histology ( Fig   1B) and the targeted analyses of TJ markers by qPCR (Fig 1C) , in that, colons of mice exposed to nicotine-free, but not nicotine-containing e-cig have impaired tight junction markers and are inflamed.

KEGG pathway (Fig 2I) and GO (Fig S3) analyses revealed that the most enriched diseaserelated pathways were those that are involved in cellular sensing and response to external stress and stimuli (peroxisome proliferator-activated receptors, PPAR and 5' AMP-activated protein kinase, AMPK signaling), cell death and programmed cell death, defense response to other organisms, metabolism (lipolysis, adipocytokine, thermogenesis) and inflammation (cytokine and receptors). With regard to cytokine signaling, we noted that il31ra (subunit for IL6R), il1r2 (decoy receptor for IL1R1), ccl8 (a chemoattractant), ear2 (chemoattractant), and ilk (activator of NFkB) were upregulated, whereas trim30a (a suppressor of NFκB) and madcam1 (a cell-adhesion molecule required for leukocyte trafficking) were downregulated. No KEGG pathways were significantly enriched among the downregulated genes. A Reactome pathway (Fig 2J) analysis on the same gene sets showed enrichment of anti-microbial peptides, specifically beta-defensins (Defb4, 6 and 14), and de-enrichment of regulators of lysosome biogenesis (sh3gl2, eya3, vamp7) and chloride transporters (slc12a3, slc12a5). Besides these statistically enriched pathways and processes, it is noteworthy that several cancer-related genes (dkkl1, trim29, s100a4, tmem45a, and klk8) were also upregulated (Fig 2B) , and as expected, an enrichment of pathways for differentiation program in the colon (e.g., multiple Keratins; Fig 2J) . Many of these differentiation-related genes continued to remain high despite nicotine (Fig S2B) . These findings suggest the upregulation of two opposing programs in the colon that tightly regulate oncogenesis in the colon.

inflammation. To translate the relevance of our findings in mice colon to the human gut, we generated organoids from colonic and ileal biopsies obtained during colonoscopy from healthy human subjects (3 independent donors, age range-29 to 71 years). These organoids were subsequently differentiated into EDMs and exposed either acutely (single exposure) or chronically (repeated exposures x 3, each 4 hours apart) prior to analyzing them at 24 hours for barrier integrity and markers of inflammation using multiple modalities (see Fig 3A) . An acute single exposure to e-cig was associated with a significant drop in TEER (-48.1±5.5%) at 4 hours (Fig 3B; left) . However, much of that initial drop at 4 hours was virtually reversed after 24 hours to levels that were similar to untreated or air-treated control EDMs (UN = 41.7±8.0%, Air = 49.1±14.5% and e-cig = 23.6±18.5% (Fig 3B; right) . When the EDMs were subjected to 3x e-cig exposures, which is a more physiologic exposure based on human use patterns, the drop in TEER was sustained at 24 hours (-59.96±3.82%; Fig 3B; right).

We also confirmed that the observed drop in TEER in the colon-derived EDMs was also associated with a loss of structural integrity of the TJs. An acute single exposure was associated with only infrequent aberrant tricellular TJ morphology and a statistically insignificant 'burst' appearance at 24 h (Fig 3C, left; Fig 3D, arrowheads) . However, repeated 3x exposures resulted in a significant ~3fold increase in the % of 'burst' TJs (Fig 3C, right; Fig 3E, arrowheads) . The levels of transcripts of the membrane-integral TJ-marker, occludin, increased after acute exposure (Fig 3F, left; Fig S4) , but returned to normal levels at 24 h (Fig 3F; right) . The levels of the peripheral TJ-marker ZO1 was unchanged at 4 h after an acute exposure (Fig 3G; left) , while there was a significant drop in gene expression of ZO1 after chronic repetitive multiple exposure (Fig 3G; right) .

Because prior studies have implicated loss of epithelial barrier integrity as permissive to inflammation (33), we next investigated inflammatory gene expression in the e-cigarette-exposed EDMs by qPCR. Compared to untreated or air-treated controls, chronic repetitive exposure to e-cigarettes (but not single acute exposure) increased expression of all pro-inflammatory cytokines tested (Fig 4A-D) .

ELISA studies conducted on EDM supernatants confirmed that pro-inflammatory cytokine IL-8 was significantly elevated (Fig 4E) .

Similar findings were also observed in the case of human ileum-derived EDMs. TEER dropped significantly at 4 hours after both single and 3x exposures (Fig S5A-C ; ~55% drop compared to control EDMs). The patterns of change in occludin and ZO1 were mirrored in the case of human ileum-derived EDMs (Fig S5D-G) . While a single acute exposure had little or no effect on cytokine transcripts (Fig   S5H, J, L, N) , chronic repetitive e-cigarette exposure caused increased IL-1B (Fig S5I) , IL-6 ( Fig   S5K) , IL-8 (Fig S5M) and MCP1 (Fig S5N) .

Taken together, these physiologic (TEER), morphologic ('burst' appearance of TJ's), and transcriptomic (qPCR assessment of markers of TJ-transcripts) readouts are all in agreement, i.e., exposure to nicotine-free e-cigarette aerosols causes epithelial barrier dysfunction in the human gut.

These data also demonstrate that chronic (repetitive), but not acute (single), exposure is necessary for such disruption. Furthermore, we found that disruption in barrier integrity in the setting of chronic repetitive exposure is also associated with the induction of pro-inflammatory cytokines.

infections. Because the gut epithelial barrier of those who vape is concomitantly exposed to chemical components of e-cigarettes as well as luminal microbes, we exposed EDMs simultaneously to both stressors. First we exposed the basolateral side of EDMs grown on transwells to e-cigarette-infused media (mimicking the absorption of core chemicals contained within nicotine-free vaping aerosols into the blood stream and diffusion into tissues) and subsequently challenged the apical surface with live pathogenic microbes (to simulate luminal microbes) (Fig 5A) . We used the adherent invasive E. coli (AIEC)-LF82, a pathogenic strain that was originally isolated from a patient with IBD (34) . Compared to untreated controls, EDMs repeatedly exposed to e-cigarette aerosol media had a significant drop in the levels of occludin mRNA (Fig 5B; left) and significant increases in the levels of transcripts of inflammatory cytokines IL-1B, IL-8 and MCP1 (Fig 5C-E) . Levels of IL-6 also trended up but fell short of statistical significance (Fig 5F) . ELISA studies confirmed that EDMs repeatedly exposed to ecigarette aerosol media also secreted higher amounts of IL-8 (Fig 5G) and MCP1 (Fig 5H) . Unlike the EDMs that were repeatedly exposed to e-cigarettes (3x), those exposed only once did not have a significant reduction in occludin (Fig 5B; left) , nor induction of proinflammatory cytokines (Fig 5C-H) .

These findings indicate that exposure to the common core chemical components of e-cigarette aerosols is sufficient to make the gut hyperreactive to microbes, and that repetitive exposure is necessary for such hyperresponsiveness.

Because cytokine production by epithelial cells after infection is a culmination of multiple events during epithelial sensing and signaling that are triggered by microbes, we next asked how exposure to ecigarettes impact some of the early steps, i.e., infectivity of the gut epithelium and epithelial reaction to such infection by production of reactive oxygen species (ROS); the latter serves as a critical second messenger which modulates innate immune signaling in the gut epithelium (35). EDMs exposed repeatedly to e-cig (3x) showed statistically significant higher number of internalized bacteria compared to control EDMs after 3 hours of infection, demonstrating decreased host defenses with higher infectivity of gut epithelium after e-cigarette exposure (Fig 5I) . Finally, we found that repeated exposures of e-cigarette aerosol media (3x) followed by infection of EDMs was associated with reduction in ROS (Fig 5J) . Unlike the chronically exposed EDMs, those exposed only once did not show a significant increase in infectivity; nor did they show a significant reduction in ROS production (Fig 5I-J) . These findings indicate that chronic e-cigarette use may lead to diminished host defenses in the gut, namely reduced ROS, leading to increased susceptibility to bacterial infections.

Taken together, these findings indicate that chronic repetitive exposure to e-cigarettes alter the gut epithelial cell response to infection with pathogenic microbes, characterized by higher infectivity and induction of proinflammatory cytokines and a failure to induce ROS. Because the overall composition of the gut microbes does not appear to be significantly altered among the subjects who consume e-cigarettes (26), our findings show that e-cigarettes may impair gut homeostasis primarily via modulation of host responses to microbes.

The major discovery we report in this work is that chronic repetitive but not acute exposure to e-cigarette aerosols disrupts the gut epithelial barrier, increases the susceptibility of the gut lining to bacterial infections and triggers gut inflammation. We also pinpoint the components in the e-liquid as the major culprit. We established causality by using near physiologic ex vivo murine and human gut models; the minimalistic nature of the enteroid monolayer system and our ability to manipulate it sequentially and in a physiologically relevant manner (context, dimension and orientation) allowed us to pinpoint the target cell for e-cig-induced injury as the gut epithelial cell. By using invasive E. coli in co-culture studies with EDMs, we also determine that handling of microbes by the gut epithelium was fundamentally impaired upon chronic and repetitive exposure to e-cig, resulting in higher infectivity and inflammation. Our findings are in keeping with prior studies showing higher infectivity and inflammation in the epithelial lining of the oral mucosa (25) and the lung (36) (37) (38) .

E-cigarettes broadly impact gut health: Other major outcomes of this study are the insights gained from the extent and nature of the altered transcriptional landscape of the murine gut that is repeatedly exposed to e-cig over months. Our RNA seq studies showed three major inter-related themes of altered transcriptional programs. First, are pathways concerning cellular response to stress and stimuli; prominent inductions of genes that participate within the PPAR and AMPK signaling pathways. Second, are pathways concerning mucosal response to infection and inflammation, with prominent induction of genes that encode the anti-microbial peptide β -defensins and downregulation of genes that concern lysosomal biogenesis and genes like Tecpr1, which is necessary and sufficient for autophagic clearance of microbes (39) . The upregulation of stress response genes and the very specific pattern of upregulation of β -isoform of defensins is not unique to the gut; transcriptomic analyses on human bronchial epithelium have documented the same previously (40) . The third and final theme is that of a balanced upregulation of genes that support pro-and anti-oncogenic pathways and processes, the most prominent of which were genes involved in cellular differentiation, i.e., multiple keratins. Because, a sufficient amount of keratin is needed for efficient stress protection in the colonic epithelia and because keratins play an essential role of maintaining epithelial barrier and its down-regulation in intestinal tissue has been correlated with the progression of IBD (41, 42) , our findings suggest that the three themes of altered gene expression may be interrelated consequences of epithelial stress response to chronic stimuli (simultaneous exposure to e-cig and microbes) and inflammation. in the blood of smokers significantly improves tight junction integrity, and thus, decreases epithelial gut permeability (43) . Similarly, studies in humans (44, 45) have shown that nicotine does tighten the gut and that such effect may only be seen upon chronic, but not acute exposures (46) (47) (48) . As for mechanism(s) behind such tightening, upregulation of TJ markers (43, 49) , most prominently that of occludin, has been reported. Others have shown that tightening of the gut barrier also translates to reduced inflammation in the gut mucosa; nicotine appears to have a direct anti-inflammatory effect on monocytes (50-53) and T cells (54, 55) , potentially through direct activation of the cholinergic antiinflammatory pathway, which involves inhibition of NFkB signaling (56) . Furthermore, nicotine has been generally found to serve as a protective factor for the development and progression of ulcerative colitis (UC), a condition that is characterized by leakiness of the gut barrier and chronic inflammation in the gut lining. That the nicotine-containing e-cig group is protected from the impacts of e-cig is most consistent with the possibility that the addition of the barrier-protective agent, nicotine, to the e-cig formulation was sufficient to most optimally balance the barrier-destructive effects of e-cig alone (showcased here). Statistical significance was estimated using one-way ANOVA with Tukey's test; ****p < 0.0001. C-E.

EDMs were treated as indicated prior to fixation, and then stained for tight junction (TJ) marker occludin (green), ZO1 (red) and DAPI (blue, nuclei) and analyzed by confocal microscopy. Bar graphs in C display the % increase in tight junction (TJ) 'bursts'. Data is displayed as mean ± (n = 3-5 fields/condition). Statistical significance was estimated using one-way ANOVA with Tukey's test; **p < 0.01; n.s. = not significant. Confocal microscopic images representative of EDMs after 24 h of treatment with either a single (D) or repeated (3x; E) exposure(s) to air (control) or nicotine-free e-cig vapor-infused media are shown. Scale bar = 10 μ m. F-G. Bar graphs display the relative fold change in mRNA expression of tight junction markers (Occludin and ZO1) in human EDMs after acute (4 h) or chronic (24 h) exposure of e-cig vapor-infused media. Data is displayed as mean ± SEM (n = 3).

Statistical significance was estimated using one-way ANOVA with Tukey's test; *p < 0.05 and ****p < 0.0001. Statistical significance was estimated using either one-way ANOVA with Tukey's test (black) or Mann-Whitney test (red); *p < 0.05, **p < 0.01 and ***p < 0.001. E. Bar graphs display the concentration of IL-8 released in the basolateral compartment of polarized EDMs after exposure to e-cig vapor-infused media. Data is shown as mean ± SEM (n = 3 independent experiments). Statistical significance was estimated using one-way ANOVA with Tukey's test; *p < 0.05 and **p < 0.01. independent experiments). Statistical significance was estimated using one-way ANOVA with Tukey's test; *p < 0.05 and **p < 0.01. I. Bar graphs display the bacterial load internalized in EDMs pretreated as indicated with or without single or repeated exposure to e-cig vapor-infused media and then exposed to pathogenic AIEC-LF82 for 3 h. Data is expressed as no. of internalized bacteria to the infected control EDMs (untreated; i.e., not exposed to e-cig) and is represented as the mean ± SEM of three separate experiments. Statistical significance was estimated using one-way ANOVA with Tukey's test; **p < 0.01. J. Bar graphs display cellular accumulation of ROS, as determined by measuring the levels of oxidized DNA in the supernatant in the basolateral compartment of polarized EDMs after exposure to the indicated treatments. Data is represented as the mean ± SEM of three separate experiments. Statistical significance was estimated using one-way ANOVA with Tukey's test;

*p < 0.05. Preparation of E-cigarette vapor-infused media: E-liquid mixture of 70% propylene glycol and 30% glycerol and 6mg/mL nicotine (purchased from Sigma) without flavors or additives were used. Ecigarette atomizer and battery were obtained from Scireq containing a Kangertech Subtank Plus (7mL), using a 0.15 ohm coil, and rechargeable battery. Fresh e-cig vapor-infused media was created by activating the battery via application of negative pressure by the InExpose system (SciReq), the e-liquid was heated and drawn through the internal atomizer and then into a 60 ml syringe containing 10 ml of wash media (DMEM/F12 with HEPES, 10% FBS). The wash media was exposed to 50mLs of e-cig vapor generated from the vaporization of the e-cig liquid, (with or without 6mg/ml nicotine) followed by a 12-second shake; this is repeated 30 times.

Human Subjects: To generate healthy enteroids, a fresh biopsy was collected from healthy subjects undergoing routine colonoscopy using the protocol approved by the Human Research Protection

Program Institutional Review Board (Project ID# 190105).

Intestinal crypts, comprised of crypt-base columnar (CBC) cells, were isolated from both colonic and ileal tissue specimens using the previously published paper (33, 57) . EDMs were generated on either 24-well or 96well transwells with a 0.4 µm pore polyester membrane (Corning) as described previously (33, 57, 58) .

The polarized differentiated EDMs were treated with e-cigarette vapor infused media, added to the basolateral compartment of the transwells for the indicated times. The EDMs were used to measure transepithelial electrical resistance (TEER), stained for immunofluorescence or the preparation of RNA and for the collection of supernatants from apical and basolateral sides. Immunofluorescence staining: After the final TEER measurement of all EDM treatments, the media was removed from apical and basolateral compartments. The exposed EDMs were fixed, permeabilized and stained for occluding and ZO-1 and imaged using a Leica TCS SP5 Confocal Microscope as done previously (33) . Z-slices of a Z-stack were overlaid to create maximum intensity projection images; all images were processed using FIJI (Image J) software.

TEER, qPCR and ELISA results were expressed as the mean ± SEM and compared using a one-way ANOVA with Tukey's test or Mann-Whitney test. Results were analyzed in the GraphPad Prism and considered significant if p-values were < 0.05.

We 

All the authors declare that they have no conflict of interest. 

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Does smoking tighten the gut?

The effects of smoking and indomethacin on small intestinal permeability

Non-smoking: a feature of ulcerative colitis

Testing nicotine gum for ulcerative colitis patients. Experience with single-patient trials

In vivo influence of nicotine on human basal and NSAID-induced gut barrier function

L-Glutamine Enhances Tight Junction Integrity by Activating CaMK Kinase 2-AMP-Activated Protein Kinase Signaling in Intestinal Porcine Epithelial Cells

Nicotine inhibits the in vitro production of interleukin 2 and tumour necrosis factor-alpha by human mononuclear cells

Nicotine inhibits cytokine synthesis by mouse colonic mucosa

Divergent effects of nicotine administration on cytokine levels in rat small bowel mucosa, colonic mucosa, and blood

An investigation into the effect and mechanisms of action of nicotine in inflammatory bowel disease

The effect of nicotine on murine CD4 T cell responses

Effects of nicotine on the immune response. II. Chronic nicotine treatment induces T cell anergy

Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation

Host engulfment pathway controls inflammation in inflammatory bowel disease

Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche

High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease

Investigation on potential associations of oxidatively generated DNA/RNA damage with lung, colorectal, breast, prostate and total cancer incidence

Experimental evidence of oxidative stress in patients with l-2-hydroxyglutaric aciduria and that l-carnitine attenuates in vitro DNA damage caused by d-2-hydroxyglutaric and l-2-hydroxyglutaric acids