Touted by makers as a “healthy” alternative to traditional nicotine cigarettes, new research indicates the chemicals found in e-cigarettes disrupt the gut barrier and trigger inflammation in the body, potentially leading to a variety of health concerns.
In the study, published Jan. 5, 2021 in the journal iScience, Soumita Das, Ph.D., associate professor of pathology, and Pradipta Ghosh, MD, professor of cellular and molecular medicine at UC San Diego School of Medicine and Moores Cancer Center at UC San Diego School of Medicine, with colleagues, found that chronic use of nicotine-free e-cigarettes led to a “leaky gut,” in which microbes and other molecules seep out of the intestines, resulting in chronic inflammation.
Such inflammation can contribute to a variety of diseases and conditions, including inflammatory bowel disease, dementia, certain cancers, atherosclerosis, liver fibrosis, diabetes and arthritis.
“The gut lining is an amazing entity. It is comprised of a single layer of cells that are meant to seal the body from the trillions of microbes, defend our immune system, and at the same time allow absorption of essential nutrients,” said Ghosh.
“Anything we eat or drink, our lifestyle choices in other words, has the ability to impact our gut microbes, the gut barrier and overall health. Now we know that what we smoke, such as e-cigarettes, negatively impacts it as well.”
The researchers found that two chemicals used as a base for all e-cigarette liquid vapor – propylene glycol and vegetable glycerol – were the cause of inflammation.
“Numerous chemicals are created when these two are heated to generate the fumes in vaping that cause the most damage, for which there are no current regulations,” said Ghosh.
“The safety of e-cigarettes have been debated fiercely on both sides. Nicotine content, and its addictive nature, has always been the major focus of those who argue against its safety, whereas lack of chemicals in the carcinogens that are present in the cigarette smoke has been touted by the makers of e-cigarettes when marketing these products as a ‘healthy alternative.’
In reality, it’s the chemicals making up the vapor liquid that we should be more concerned about as they are the cause of gut inflammation.”
For the study, the team used 3-D models of human intestinal tracts generated from patient cells and simulated what happens when e-cigarette vapors enter the gut lining. Researchers validated the findings using mice models of vaping in collaboration with Laura Crotty-Alexander, MD, associate professor of medicine in the Division of Pulmonary, Critical Care and Sleep Medicine at UC San Diego School of Medicine and section chief of Pulmonary Critical Care at Veterans Affairs San Diego Healthcare System.
To produce the 3-D gut organoids, the researchers collected stem cells from patients’ biopsies during colonoscopies and grew them in vitro. The stem cells differentiated into the four different cell types that make up the gut lining. The team then exposed the organoids to e-cigarette liquid vapor, mimicking the frequency of a chronic vaper.
They noted that epithelial tight conjunction markers, which are zipper-like proteins that form the gut’s first physical barrier, began to break or loosen, causing pathogens from the vapor to seep into the surrounding immune system, wreaking havoc on protective epithelial cells that lie just beneath.
Such cells act as a defense against infection by clearing pathogenic microbes and initiating certain immune responses in the body. When exposed to the e-cigarette liquid, the cells were quickly overwhelmed, unable to effectively clear pathogens, resulting in gut inflammation.
The study is part of the HUMANOID Center of Research Excellence, a core facility based at UC San Diego School of Medicine led by Ghosh and Das who was senior author of the study. Scientists at the center use a variety of human organoids and other tools to model diseases and effects.
“This is the first study that demonstrates how chronic exposure to e-cigarettes increases the gut’s susceptibility to bacterial infections, leading to chronic inflammation and other health concerns,” said Das. “Given the importance of the gut barrier in the maintenance of the body’s immune homeostasis, the findings provide valuable insight into the potential long-term harmful effects chronic use of e-cigarettes on our health.”
Ghosh said damage to the gut lining may be reversible over time if the inciting factor, in this case e-cigarette use, is eliminated, but the effects of chronic inflammation upon other organs, such as the heart or brain, may be irreversible. In the future, Ghosh said she and colleagues plan to look at different flavorings of e-cigarettes to determine what effects they might have on the gut.
Daily e-cigarette aerosol inhalation drives inflammation in the colon and reduces the expression of genes related to barrier function.
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 (1 h/day) at two time-points: 1 week (resembling acute exposure) and 3 months (resembling chronic exposure) (Figure 1A).
Because the most common chemicals in e-cigarette aerosols are nicotine and humectants (propylene glycol and vegetable glycerin), we utilized nicotine-free and nicotine-containing (6 mg/ml) e-liquids with a 70:30 ratio of propylene glycol and vegetable glycerol (PG:VG) within a Kanger Subtank attached to a box Mod e-device, and used exposure chambers with room air for controls (Figure 1A).
This low concentration of nicotine (6 mg/ml) was selected on the basis of several published works (ranging typically between ~6 – 9 mg/ml) and the amount present in the most popular brands (Cox et al., 2016; Dawkins et al., 2016; Stewart et al., 2018).
The ratio of 70:30 PG:VG was chosen because it is the most commonly used and preferred ratio ratio as per consumer experience (Smith et al., 2020). H&E staining of distal colons from mice acutely exposed to nicotine-free (vehicle only) e-cigarette aerosols (e-cig) for 1 week demonstrated small, infrequent patches of leukocyte infiltration in the submucosal layers (Figure 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 over 3 mon led to large submucosal inflammatory infiltrates within the colon (Figure 1B, right). No inflammatory infiltrates were seen in air controls, and only smaller and infrequent infiltrates were present in colons of mice chronically exposed to nicotine-containing e-cig (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 (Figure 1C). The fact that e-cig exposure affects the levels of Claudin-2, a major regulator of TJ-specific obliteration of the intercellular space (Kubota et al., 1999), but not its counterpart Claudin-1 (Figure 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 (Figure 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 (Figure S1).
These findings indicate that chronic, but not acute exposure to aerosols of nicotine-free e-cigarettes 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 ameliorate both phenotypes.
E-cigarettes disrupt the integrity of the gut barrier.
An intact gut barrier is an important first-line defense. To determine if the observed decrease in colon TJ markers in mice exposed to e-cigs is a direct consequence of circulating chemicals inhaled in the aerosols on the gut epithelial barrier, we used an ex vivo near-physiologic model system called the “gut-in-a-dish” (Figure 1D) (Ghosh et al., 2020).
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). The EDMs are also widely believed to be a model that is superior to cultured colon cancer cell lines (e.g., Caco2) because –
- (i) they 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 (Foulke-Abel et al., 2014; Mahe et al., 2013; Miyoshi and Stappenbeck, 2013; Noel et al., 2017);
- (ii) they are not transformed and yet allow culturing over several passages so that they can be used to ensure reproducibility of meaningful functional studies;
- (iii) because they preserve dimentionality, i.e., apicobasal polarity with functional tight junctions that can recreate the gut barrier ex vivo.
To dissect how e-cig-derived chemicals in the systemic circulation impact the gut barrier, we exposed the basolateral surfaces of the murine colonic EDMs to e-cigarette aerosol-infused media. As negative controls, we either exposed the EDMs to normal growth media (‘UN’) or to air-infused media; (‘Air’). We analyzed the integrity of the gut barrier using two readouts (Figure 1D):
- (i) paracellular permeability, as reflected by low trans-epithelial electrical resistance (TEER)
- (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 (Figure 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 (Figure 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 (Furuse et al., 2014), are also the regions where TJ-disruption can be visualized/assessed first (Ghosh et al., 2020)] by ~60.1±8.1% when compared to untreated (p = 0.0010) and air-treated (p = 0.0015) controls (Figure 1F-G).
These findings show that chemicals contained within the most basic e-cigarette aerosols have a disruptive effect on the epithelial barrier.
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.
Chronic exposure to inhaled e-cigarette aerosols induces stress-responses in the colon.
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 (Figure S2A), chronic exposure was associated with significant changes (Figure 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) (Figure 2B; Table S1). Barring a handful of genes (arrowheads, Figure 2B, S2B), most of these differences were abolished when mice were exposed to nicotine-containing e-cigarettes (Figure 2B). TJ markers occludin and ZO1 were downregulated by nicotine-free, but not nicotine-containing e-cigarettes (Figure 2C-D).
Multiplepro-inflammatory cytokines were either elevated significantly (MCP1, IL-8, TNFα) or showed an increasing trend but did not reach significance (Cxcl2) (Figure 2E-H). These RNA Seq findings are in agreement with our prior observations by histology (Figure 1B) and the targeted analyses of TJ markers by qPCR (Figure 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 (Figure 2I) and GO (Figure S3) analyses revealed that the most enriched disease- related 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 NFkB) 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 (Figure 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 (Figure 2B), and as expected, an enrichment of pathways for differentiation program in the colon (e.g., multiple Keratins; Figure 2J).
Many of these differentiation-related genes continued to remain high despite nicotine (Figure S2B). These findings suggest the upregulation of two opposing programs in the colon that tightly regulate oncogenesis in the colon.
Chronic (repetitive) exposure to e-cigarettes disrupts the integrity of the human gut barrier and triggers inflammation.
To translate the relevance of our findings in mice colon to the human gut, we generated organoids from colonic and ileal biopsies obtained 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 Figure 3A).
An acute single exposure to e-cig was associated with a significant drop in TEER (-48.1±5.5%) at 4 hours (Figure 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% ( Figure 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%; Figure 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 (Figure 3C, left; Figure 3D, arrowheads).
However, repeated 3x exposures resulted in a significant ~3-fold increase in the % of ‘burst’ TJs (Figure 3C, right; Figure 3E, arrowheads). The levels of transcripts of the membrane-integral TJ-marker, occludin, increased after acute exposure (Figure 3F, left; Figure S4), but returned to normal levels at 24 h (Figure 3F; right). The levels of the peripheral TJ-marker ZO1 was unchanged at 4 h after an acute exposure (Figure 3G; left), while there was a significant drop in gene expression of ZO1 after chronic repetitive multiple exposure (Figure 3G; right).
Because prior studies have implicated loss of epithelial barrier integrity as permissive to inflammation (Ghosh et al., 2020), 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 transcripts for all pro-inflammatory cytokines tested (Figure 4A-D). ELISA studies conducted on EDM supernatants confirmed that the protein levels of the pro-inflammatory cytokine IL-8 were significantly elevated (Figure 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 (Figure 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 (Figure S5D-G). While a single acute exposure had little or no effect on cytokine transcripts (Figure S5H, J, L, N), chronic repetitive e-cigarette exposure caused increased IL-1B (Figure S5I), IL-6 (Figure S5K), IL-8 (Figure S5M) and MCP1 (Figure 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. They also demonstrate that chronic (repetitive), but not acute (single), exposure is necessary for such disruption and that such disruption is associated with the induction of pro-inflammatory cytokines.
Chronic (repetitive) exposure of the gut epithelium to e-cigarette aerosols accentuates inflammatory responses to 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) (Figure 5A).
As before (in Fig 3), EDMs were either treated acutely (single exposure) or chronically (repeated exposures x 3, each 4 hours apart) with e-cig-infused media (ECIG X3). In the negative controls, that were exposed only to media (UN), the media was changed every 4 hours (UNX3), just like the e-cig condition. We used the adherent invasive E. coli (AIEC)–LF82, a pathogen that was originally isolated from a patient with IBD (Boudeau et al., 1999).
Compared to untreated controls, EDMs repeatedly exposed to e-cigarette aerosol media had a significant drop in the levels of occludin mRNA (Figure 5B; left) and significant increases in the levels of transcripts of inflammatory cytokines IL-1B, IL-8 and MCP1 (Figure 5C-E). Levels of IL-6 also trended
up but fell short of statistical significance (Figure 5F). ELISA studies confirmed that EDMs repeatedly exposed to e-cigarette aerosol media also secreted higher amounts of IL-8 (Figure 5G) and MCP1 (Figure 5H). Unlike the EDMs that were repeatedly exposed to e-cigarettes (3x), those exposed only once did not have a significant reduction in occludin (Figure 5B; left), nor induction of proinflammatory cytokines (Figure 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 an infection is a culmination of multiple events during epithelial sensing and signaling that are triggered by microbes, we next asked how exposure to e- cigarettes 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 (Jones et al., 2012).
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 (Figure 5I).
Finally, we found that repeated exposures of e-cigarette aerosol media (3x) followed by infection of EDMs were associated with a reduction in ROS (Figure 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 (Figure 5I-J).
Taken together, these findings indicate that chronic repetitive exposure to e-cigarettes alters the gut epithelial cell response to infection with pathogenic microbes, characterized by higher infectivity and induction of pro-inflammatory cytokines and a failure to induce protective ROS. Because the overall composition of the gut microbes does not appear to be significantly altered among the subjects who consume e-cigarettes (Stewart et al., 2018), our findings show that e-cigarettes may impair gut homeostasis primarily via modulation of host responses to microbes.
reference link : doi: https://doi.org/10.1016/j.isci.2021.102035.
More information: E-cigarettes compromise the gut barrier and trigger inflammation, iScience, DOI: 10.1016/j.isci.2021.102035 , www.cell.com/iscience/fulltext … 2589-0042(21)00003-1
