Selective Activation of Human Monocytes Exposed Ex Vivo to Different E-Cigarette Aerosols: Possible Role in Subclinical Inflammation

Highlights

What are the main findings?

• Electronic cigarette aerosols, especially selected flavors, selectively activate small subsets of human monocytes, increasing adhesion markers, oxidative stress, TNFα, and CD68 expression while only modestly affecting whole-population cytotoxicity compared with cigarette smoke.
• These aerosols markedly reduce phagocytic activity and drive selected monocytes into highly oxidative/pro-inflammatory subsets that resemble macrophage-like, CD68-high cells, with effects largely attributable to non-nicotine aerosol constituents.

What are the implications of the main findings?

• Vaping, though less globally toxic than cigarette smoke, can sustain subclinical inflammation by promoting adhesion-prone, pro-oxidative, TNFα-rich monocyte subsets that may preferentially accumulate at sites of vascular or tissue injury.
• Flavor-dependent monocyte activation suggests that certain e-cigarette products may carry underestimated long-term cardiopulmonary risk, particularly in chronic users, dual users, or individuals with pre-existing inflammatory or cardiovascular disease.

Abstract

Electronic cigarettes (ECs) are promoted as a safer alternative to traditional cigarettes, yet their impact on immune cells remains incompletely understood. This study investigates the activation of human primary adherent and non-adherent monocytes exposed ex vivo to aerosols from four flavored ECs (classic tobacco, menthol, watermelon, and strawberry) compared to cigarette smoke (CS) and nicotine alone. EC aerosols (ECEs) induced modest cytotoxicity, oxidative stress, and superoxide dismutase activity compared to CS, with high cell response heterogeneity indicating subpopulation-specific effects. Adherent monocytes showed elevated integrin expression (CD11a, CD11b), ICAM-1 (CD54), TNFα, and oxidative stress versus non-adherent cells, amplified by ECE. Dual fluorescence flow cytometry (green DCF for ROS and red for anti-TNFα Ab) revealed shifts toward pro-inflammatory/oxidative quadrants, particularly upper-right high-intensity relatively small subsets with macrophage M1-like CD68 expression in adherent cells. ECEs reduced phagocytosis in adherent monocytes, mimicking CS effects, probably driven by non-nicotine components. Strawberry flavor (ECE 4) elicited the strongest TNFα induction. These findings demonstrate EC-induced subclinical inflammation via selective monocyte activation, potentially contributing to chronic cardiopulmonary risks despite significantly lower overall toxicity than CS.

1. Introduction

E-cigarettes (EC) are promoted as a healthier alternative to traditional cigarettes. They have gained popularity, but their impact on public health is still unknown. EC aerosols are much less toxic than traditional cigarettes in in vitro assays [1], but both e-liquids and exposures are also more diverse. There are no reports that EC use is directly related to lung diseases, but experimental and clinical studies have shown that EC “vaping” can cause a variety of respiratory symptoms and produce negative effects on existing lung diseases [2,3,4]. A 2024 review by Glanz et al. [5] estimated that ECs were one-third as harmful as regular cigarettes. The authors concluded that there is a need to reassess the assumption that EC use provides substantial harm reduction across all cigarette-caused diseases, particularly accounting for dual use. Now, a growing number of studies provide evidence that e-liquids and their aerosols can be cytotoxic and cause respiratory inflammation [6,7]. E-liquid is typically a mixture of propylene glycol, vegetable glycerin, nicotine, and a wide range of flavoring chemicals, which vary between brands [7,8]. Many of these flavors are considered safe to ingest, but there are no data on chronic inhalation exposure. Some flavors have established respiratory toxicity [8], and their negative impact on the respiratory tract has been confirmed in preclinical and clinical studies [9,10]. EC-derived acrolein and formaldehyde are clearly carcinogenic and cardiotoxic [11,12]. The results of experimental and clinical studies are highly variable due to differences in chemical composition and exposures, different physical devices, and users’ health. Moreover, vaping increases the frequency of viral and bacterial infections by affecting epithelial cells, neutrophils, and alveolar macrophages [13]. Most published data show that ECs activate inflammation and increase oxidative stress [14,15]. Both processes are interrelated and may produce exacerbations in patients with lung diseases.

Monocytes and macrophages play a critical role in inflammation and oxidative stress and are key components of the immune system [16]. Activated macrophages release pro-inflammatory cytokines, especially tumor necrosis factor (TNFα), nuclear factor-κB (NF-κB), and other reactive molecules, leading to the overproduction of reactive oxygen species (ROS) [17,18]. It was shown that EC increased a key pro-inflammatory cytokine, TNFα, in monocytes and activated selected biochemical pathways of monocyte–macrophage transition [19,20,21,22]. This response, however, is not fulminant and probably involves only selected cells. Anti-oxidative and pro-inflammatory effects were also described in monocyte-macrophage cell line with ECE, CS and other nicotine delivery systems by all products [19]. Cell scatter flow cytometry allows for the quantitative analysis of the restrained effects of EC aerosols. Therefore, this work aims to assess the effect of four popular EC on the activation of TNFα, changes in intracellular redox status, and co-expression of integrin and phagocytosis in cultured human monocytes to facilitate further comparative studies and categorize EC according to toxicity.

2. Materials and Methods

2.1. Reagents

All chemicals used in this study were purchased from Sigma Chemical Co. (Poznan, Poland) unless otherwise stated, while culture media and cell culture reagents were obtained from GIBCO (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescent antibodies were sourced from Abcam (Cambridgeshire, UK), Cell Signaling Technology (Danvers, MA, USA), and Santa Cruz Biotechnology (Dallas, TX, USA).

2.2. Cell Cultures and Treatment

Human primary monocytes were used in this study. Peripheral blood mononuclear cells were isolated from healthy blood donors of both sexes, and monocytes were purified from blood or buffy coats using CD14+ magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany). All procedures were approved by the Ethics Committee of the Medical University of Bialystok (decision APK.002/138/2022 from 24.03.2022). Monocytes were cultured in ATCC-formulated RPMI 1640 medium supplemented with 0.05 mM 2-mercaptoethanol, 2 mM L-glutamine, 5% FCS, and 100 U/mL of PenStrep. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. For experiments, cells were seeded in 6- or 12-well plates, incubated overnight in serum-free medium, and subsequently exposed for 24 h to one of the following: control medium, e-cigarette aerosol-conditioned medium (flavors: classic tobacco, menthol, watermelon, strawberry), cigarette smoke-conditioned medium, or N-supplemented serum-free medium.

The CS and EC-conditioned media were prepared using a fixed puffing regimen. Aerosol was generated from P1-brand e-liquids (CHIC Sp. z o.o, Ostrzeszow, Poland; 12 mg/mL of nicotine) in the four most popular flavors chosen to represent a range of e-cigarette categories and compositions. The aerosol was drawn through culture medium using a low-pressure vacuum pump, with settings adjusted to yield aerosol generation times similar to those for regular cigarettes. The puff duration was 3 s, with 10 s intervals. Puff volume was not measured. All exposures were performed using the same pump, operated under identical settings, which generated a stable negative pressure during each puff. The same setup was applied to both combustible cigarettes and electronic cigarettes. CS-conditioned medium was prepared from two Marlboro Red cigarettes (Philip Morris, Krakow, Poland; full strength, filters removed); smoke was passed through culture medium using a low-pressure vacuum pump set to approximately 1 min per cigarette, and the resulting medium was sterilized via filtration through 0.22 µm filters. Under these conditions, one combustible cigarette was consumed after 12 puffs. Following exposure, the nicotine content in the cigarette smoke-conditioned medium was determined and was set to approximately 0.1 mg/mL, as assessed using GC–MS analysis [23]. For electronic cigarettes, the equimolar nicotine concentration in the conditioned medium was achieved after 6 puffs; this puff number was therefore used to generate aerosol-conditioned media for subsequent experiments. Exposure conditions were thus normalized to nicotine concentration rather than puff number, enabling direct comparison between cigarette smoke- and e-cigarette aerosol-conditioned media.

Cell Exposure

All conditioned media were sterilized using 0.22 µm filters and immediately applied to cell cultures. For each experiment, cells were seeded in 6-, 24-, or 96-well plates and incubated for 24 h in either control or conditioned media.

2.3. Microscopy and Cell Morphology

Following 24 h of incubation in the appropriate conditioned medium, Giemsa-Wright staining was performed. Cells were then examined under a light microscope to evaluate morphology and cell proliferation under each experimental condition.

2.4. Cell Viability Test

Cell viability was assessed using the trypan blue exclusion assay on cyto-spin preparations or culture dishes. In this method, trypan blue selectively stains non-viable cells blue, while viable cells remain unstained. Cell suspensions were mixed 1:1 with 0.4% trypan blue; non-adherent (floating) cells were collected and centrifuged onto slides, whereas adherent cells were counted directly using an inverted light microscope. Viability was then expressed as the percentage of dead (trypan blue-positive) cells.

2.5. Assessment of Oxidative Stress

Intracellular oxidative stress was quantified using the DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) assay, a widely used green fluorescent probe for detecting global ROS generation in living cells. Upon entering cells, non-fluorescent DCFDA is deacetylated by intracellular esterases to the reduced form H2DCF, which is subsequently oxidized by ROS to highly fluorescent dichlorofluorescein (DCF), with signal intensity proportional to the intracellular oxidative burden. DCFDA-derived fluorescence was recorded as single-parameter histograms, and these distributions were used to assess treatment-induced changes in cellular redox status. In this analysis, a rightward shift of the DCF fluorescence peak along the intensity axis, together with an increase in mean and/or median fluorescence intensity, was interpreted as an indicator of elevated ROS levels and enhanced oxidative stress within the cell population. Conversely, overlapping histograms with comparable average intensities were taken to reflect unchanged basal ROS production relative to untreated controls. To validate assay performance and confirm that the experimental system was capable of mounting a detectable oxidative response, cells were exposed to tert-butyl hydroperoxide (tBHP), a well-established chemical inducer of ROS, at a final concentration of 50 µM, which served as a positive control. Under these control conditions, tBHP consistently produced a marked rightward shift in DCF fluorescence and a robust increase in average signal intensity, thereby verifying the responsiveness and dynamic range of the readout and supporting the interpretation of treatment-induced fluorescence changes as genuine oxidative stress signals.

2.6. Specific Cu/Zn Superoxide Dismutase Activity

Cu/Zn superoxide dismutase (SOD) activity was determined using the Superoxide Dismutase Activity Assay Kit (Sigma-Aldrich, CS0009, St. Louis, MO, USA; Merck Life Science Sp., Poznań, Poland), following the manufacturer’s instructions.

2.7. Quantification of Markers of Cell Adherence: CD11a, CD11b and CD54 and CD68

Cells were prepared as described above, then incubated in a blocking solution containing 5% normal mouse serum, 5% normal rat serum, and 1% FcBlock (eBiosciences, San Diego, CA, USA) in PBS, followed by staining with a standard panel of immunophenotyping monoclonal fluorescent antibodies against CD11a, CD11b, CD54, and CD68, along with the corresponding isotype controls, for 30 min at room temperature. After staining, the cells were washed twice with PBS and fixed in 0.4% paraformaldehyde. Flow cytometric data were acquired on a Beckman Coulter CytoFlex cytometer (Beckman Coulter, Warsaw, Poland) and analyzed using Kaluza software (version 2.1.2).

2.8. Quantification of Intracellular TNFα

Cells were treated as described previously, fixed for 10 min in 4% methanol-free formaldehyde at room temperature, and subsequently stained with an anti-TNFα antibody conjugated to peridinin chlorophyll protein–cyanine5.5 (PC5.5; Cell Signaling Technology, Danvers, MA, USA) diluted 1:500 in a permeabilization/wash buffer containing 0.1% Triton X-100 (Sigma Chemical Company, Poznań, Poland) and 1% bovine serum albumin. Samples were incubated for 20 min at room temperature in the dark, then washed and passed through a 100 µm cell strainer (Fisher Scientific, Poznań, Poland) to remove cell aggregates. A total of 5000 cells per sample were acquired on a Beckman Coulter CytoFLEX flow cytometer (Beckman Coulter, Warsaw, Poland), with doublets excluded by forward scatter (FSC) area versus height on a linear scale. TNFα expression was assessed as a rightward shift in red fluorescence intensity histograms relative to negative controls. As a positive control, cells were stimulated with 100 ng/mL of lipopolysaccharide (LPS from E. coli O111:B4, Sigma-Aldrich L2630, St. Louis, MO, USA) for 24 h.

2.9. Phagocytosis Assays

Monocytes were prepared as described above, washed with washing buffer, and incubated for 2 h with latex beads from the Cayman Phagocytosis Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) that were coated with PE-conjugated rabbit IgG. Following incubation, cells were washed twice with washing buffer, and internalized fluorescent beads were quantified via flow cytometry using a Coulter CytoFlex instrument (Beckman Coulter, Warsaw, Poland).

2.10. Oxidative Stress vs. TNFα Scatterplots and CD68 Expression

Intracellular oxidative stress and TNFα were visualized simultaneously on binary scatter plots to characterize ECE-induced, subgroup-specific pro-oxidative and pro-inflammatory responses of monocytes in culture. CD68 expression was included as an additional marker to compare typical macrophage-associated epitope levels, enabling assessment of monocyte activation status within clearly defined monocyte subpopulations. Following culture, cells were fixed for 10 min at room temperature in 4% methanol-free formaldehyde and subsequently stained by direct labeling with a TNFα-specific rabbit monoclonal antibody (1:200) conjugated to peridinin chlorophyll protein–cyanine5.5 (PC5.5; Cell Signalling, Danvers, MA, USA) together with DCFDA to detect intracellular reactive oxygen species. Staining was performed in permeabilization/wash buffer containing 0.1% Triton X-100 (Sigma Chemical Company, Poznan, Poland) and 1% bovine serum albumin to ensure adequate membrane permeabilization and to minimize nonspecific binding. In selected experimental conditions, an anti-CD68 monoclonal antibody conjugated to Alexa Fluor 750 (Cell Signalling, Danvers, MA, USA) was added to enable parallel quantification of CD68 in specific monocyte subsets. Samples were incubated for 20 min at room temperature in the dark, washed to remove unbound reagents, and passed through a 100 µm cell strainer (Fisher Scientific, Poznan, Poland) to eliminate aggregates before acquisition. A total of 5000 cells per sample were analyzed on a Beckman Coulter CytoFlex flow cytometer (Beckman Coulter, Warsaw, Poland), with doublets excluded manually by plotting the forward scatter (FSC) signal area versus FSC signal height on a linear scale. Green and red fluorescence channels were used to record DCFDA and TNFα signals, respectively, and the resulting data were displayed as histograms, bivariate cytograms, and fluorescence density plots to visualize shifts in signal intensity and population distribution. Flow cytometry data were further evaluated for distribution, central tendency (median/mean fluorescence intensity), and vector characteristics using Kaluza Software (version 2.1.2), providing a quantitative description of ECE-induced changes across monocyte subgroups. Far-red fluorescence from CD68 was also measured and compared between defined subpopulations to relate oxidative and inflammatory readouts to phenotypic markers of monocyte/macrophage activation.

2.11. Statistical Analysis

Statistical analyses were conducted to evaluate differences within and between experimental groups. For single-group analyses, data were compared with the hypothetical mean (or reference value) using a one-sample Student’s t test, applied to normally distributed continuous variables. For comparisons involving more than two groups, a one-way analysis of variance (ANOVA) was used, followed by Bonferroni’s post hoc multiple-comparison procedure to adjust for repeated testing and control the family-wise type I error rate. A two-sided significance level of p < 0.05 was adopted for all inferential tests, and results with p values below this threshold were interpreted as statistically significant. All statistical tests were performed as two-tailed to detect both increases and decreases relative to the comparator.

3. Results

3.1. Cell Viability, Oxidative Stress and SOD Activity

Figure 1 presents the impact of four electronic cigarette extracts: ECE 1 (classic tobacco), ECE 2 (menthol), ECE 3 (watermelon), ECE 4 (strawberry), cigarette smoke (CS), and nicotine (N) on cell viability (A), oxidative stress (B), and superoxide dismutase (SOD) activity (C) in human monocytes. CS caused a statistically significant increase (p < 0.01) in all three parameters, while the other treatments produced only minor or non-significant alterations. Regarding oxidative stress, a small increase was observed after ECE 2 (p < 0.05) and ECE 4 (p < 0.05), compared with nicotine-treated cells (p < 0.05 for both groups). Interestingly, the standard deviation in ECE-treated cells was about three times higher than in control or nicotine-treated cells, indicating considerable heterogeneity in cell response. SOD was increased (p < 0.01) only in CS-treated cells.

3.2. Cytotoxicity, Oxidative Stress, Phagocytosis and Inflammation in Adherent and Non-Adherent Monocytes

Table 1. The effect of electronic cigarette extracts: ECE 1 (classic tobacco), ECE 2 (menthol), ECE 3 (watermelon), ECE 4 (strawberry), cigarette smoke (CS) and nicotine (N) on cell adhesion, expression of integrin 11a (CD11a), integrin 11b (CD11b), intracellular adhesion molecule 1 (CD54), intracellular TNFα, oxidative stress and phagocytosis in floating (MF) and adherent monocytes (MA). CD11a, CD11 b, CD54 and intracellular TNFα were quantified in separately non-adherent and adherent cells using fluorescent monoclonal antibodies and flow cytometry detection, as described in Materials and Methods. Oxidative stress was assessed with fluorescent probe 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate staining, while phagocytosis was quantified using fluorescent latex beads and flow cytometry detection. All values are relative and refer to control monocytes expressed as 100% from 5–6 experiments.

Cell Adhesion, Integrins, TNFα, Oxidative Stress, Phagocytosis
Control		ECE 1	ECE 2	ECE 3	ECE 4	CS	N
Cell adhesion (% of adhering cells)
MA	28 ± 8	43 ± 11 *′	41 ± 9 *′	38 ± 10	42 ± 10 ′	15 ± 19	27 ± 5
CD11a (relative units)
MF	100 ± 34	124 ± 26	131 ± 32	132 ± 38	120 ± 37	178 ± 51 *′	112 ± 32 ^
MA	143 ± 31	253 ± 66 **′##	192 ± 31 *	187 ± 37 #	255 ± 67 **##′	224 ± 71 *	145 ± 50
CD11b (relative units)
MF	100 ± 42	133 ± 29 ^	136 ± 65	111 ± 15 ^^	115 ± 35 ^^	221 ± 53 **	117 ± 31 ^^
MA	177 ± 51 #	242 ± 63 ##	266 ± 57 *′	255 ± 38 *##′	213 ± 56 ##^	312 ± 72 **″	179 ± 40 ##^^
CD54 (relative units)
MF	100 ± 23	105 ± 21 ^^	165 ± 41 *^^	178 ± 46 *^^	154 ± 47 ^^	256 ± 74 **	177 ± 49 *^^
MA	199 ± 70 #	221 ± 73 ##^	352 ± 72 **′	311 ± 52 **##′	298 ± 66 **##′	343 ± 92 **′	201 ± 68 ^
TNFα (relative units)
MF	100 ± 24	111 ± 41 ^^	151 ± 39 *^^′	178 ± 46 *^^′	187 ± 67 **″	272 ± 54 **″	119 ± 33 ^^
MA	145 ± 61 #	166 ± 46 ##	169 ± 51 **	217 ± 59 ##	232 ± 73 *	371 ± 95 **	175 ± 59 *^^
Oxidative stress (relative units)
MF	100 ± 53	145 ± 71 ^^	144 ± 75 ^^	109 ± 53 ^^	137 ± 54 ^^	531 ± 91 **″	133 ± 35 ^^
MA	148 ± 72 ##	311 ± 82 *##^^′	346 ± 71 **^^″	258 ± 74 *##^^	292 ± 63 *##^^′	584 ± 96 **^^″	1595 ± 45 *^^
Phagocytosis (relative units)
MF	100 ± 33	74 ± 32	112 ± 38 ′^	66 ± 29 ′	58 ± 44 ′	49 ± 34 *′	86 ± 24 ^
MA	147 ± 32 #^^”	96 ± 37 *##^	78 ± 33 **##	85 ± 31 **##^	87 ± 47 **#	86 ± 28 **#″	89 ± 23 *^^

* p < 0.05, ** p < 0.01 for comparisons with the corresponding control cells. # p < 0.05, ## p < 0.01 for comparisons between MF and MA. ^ p < 0.05, ^^ p < 0.01 for comparisons with CS-treated cells. ′ p < 0.05, ″ p < 0.01 for comparisons with N-treated cells.

shows the effect of ECE 1–4 on adhesion, integrin expression, intracellular TNFα, oxidative stress and phagocytosis in floating (MF) and adherent (MA) monocytes. The effect of cigarette smoke (CS) and nicotine (N) is also shown.

About 28% of control M was found adherent, and three ECS significantly increased numbers of adherent cells, maximally to 44% of total cell numbers (p < 0.05), except for ECE 3, where numbers of adherent cells were slightly higher than control but not significant. MA had higher expression of integrin 11b (CD11b; p < 0.05), higher intercellular adhesion molecule 1 (CD54; p < 0.05), higher levels of tumor necrosis factor alpha (TNFα; p < 0.01) and increased oxidative stress (p < 0.01), comparing to the floating cells, but similar integrin 11a (CD11a) levels and phagocytosis. All ECS, including ECE 3, significantly increased expression of CD11a, CD11b and CD54 by about twice in MA, compared to MF. The effects of CS or N were not significant, except that N elevated CD11b by 52% (p < 0.01). The differences between distinctive ECS were generally not significant, considering adhesion markers, while significant differences were observed in TNFα content in MF. ECE 4 increased TNFα in MA by 2.5-fold (p < 0.01) and by more than almost two-fold (p < 0.01) in MF. Other ECEs provoked more moderate effects, however, always more evident in adhering cells. Concerning oxidative stress, DCF fluorescence was again more intense in adherent cells, but not significant due to high standard deviation. Significant variations were also observed in FM exposed to ECE, with a tendency toward increased oxidative stress, while in AM, all ECE significantly elevated DCF fluorescence. CS produced about five-fold increases in both AM and FM (p < 0.01), and nicotine did not have an effect. There was a significant increase (p < 0.05) in phagocytosis in MA vs MF, but pretreatment of cells with ECEs did not affect MF phagocytosis, but decreased MA phagocytosis. CS produced a important decrease in phagocytosis, by 51% (p < 0.05) and 57% (p < 0.01), for MF and MA, respectively, while nicotine alone was without significant effect, but slightly lower values in MF and MA were observed.

3.3. Double Fluorescence Analysis of Adherent and Non-Adherent Monocytes

Table 2. Quantitative analysis of dual fluorescence was performed in non-adherent (MF) and adherent (MA) monocytes exposed to ECE 1 (classic tobacco), ECE 2 (menthol), ECE 3 (watermelon), ECE 4 (strawberry), cigarette smoke (CS) or nicotine (N), followed by staining with the green fluorescent probe H2DCFDA to detect oxidative stress and a red fluorescent monoclonal antibody specific for TNFα to assess inflammatory activation. Double-labeled cells were acquired on a Beckman Coulter CytoFlex flow cytometer and visualized as bivariate fluorescence density plots, enabling characterization of the relationship between oxidative stress (green channel) and the pro-inflammatory cytokine TNFα (red channel) at the single-cell level. For analytical purposes, each plot was arbitrarily partitioned into four quadrants: lower left (LL, low green/low red), lower right (LR, high green/low red), upper left (UL, low green/high red) and upper right (UR, high green/high red). Quadrant thresholds were established using untreated control cells to obtain an initial distribution of 25% of events per quadrant. In adherent monocytes, the UR quadrant was further subdivided into UR L and UR H regions, corresponding to relatively lower and higher fluorescence intensities, respectively. Slopes of symmetry axes within UR L and UR H subpopulations, as well as the CD68 expression ratio in URH versus URL cells, are shown. Data are presented as means ± SD; all samples were analyzed in triplicate, and the experiment was independently replicated three times.

Oxidative Stress/TNFα Scatterplots
(%)	Control	ECE 1	ECE 2	ECE 3	ECE 4	CS	N
MF
UL	25 ± 7	21 ± 4 ″^^	16 ± 6	19 ± 5 ^	16 ± 5 *	12 ± 5 **″	21 ± 6 ^^
UR	25 ± 6	33 ± 7 ^	31 ± 7 ^^	37 ± 7 *′	41 ± 8 **^″	33 ± 9	27 ± 5
LL	25 ± 5	18 ± 3 *^^′	23 ± 3 ^^	18 ± 5 *^^″	9 ± 3 **^^″	6 ± 5 **″	26 ± 6 ^^
LR	25 ± 7	28 ± 6 ^^″	32 ± 6 ^^	26 ± 4 ^^	34 ± 5 *″	49 ± 11 **″	26 ± 4 ^^
MA
UL	25 ± 4	17 ± 4 *	26 ± 7 #^^″	16 ± 5 *	24 ± 4 #^^	8 ± 5 **″	24 ± 6 #^^
UR	25 ± 6	43 ± 6 **#″	35 ± 7 **	45 ± 5 **#″	41 ± 5 **#^^′	42 ± 7 **##′	31 ± 6 **^
UR L		34 ± 8	24 ± 6	32 ± 8	26 ± 9	26 ± 8	22 ± 7
UR H		9 ± 2 **#	11 ± 2 **	13 ± 5 **	15 ± 5 **	16 ± 6 **	9 ± 3 **#
LL	25 ± 5	9 ± 3 **##″	11 ± 4 **##″	8 ± 4 **##″	6 ± 4 **″	6 ± 4 **″	19 ± 4 ^^
LR	25 ± 4	31 ± 7	28 ± 5	31 ± 6	29 ± 6	44 ± 7 ##	26 ± 5 ^^
URH/URL
CD68	1.0 ± 0.12	1.33 ± 0.21 *^	1.24 ± 0.22 ^^	1.44 ± 0.25 **	1.48 ± 0.27 **	1.79 ± 0.31 **	1.17 ± 0.18 ^^
UR L slope	0.58 ± 0.1	0.57 ± 0.2	0.56 ± 0.1	0.51 ± 0.1	0.47 ± 0.1	0.45 ± 0.2 *	0.54 ± 0.2
UR H slope	0.87 ± 0.2	0.92 ± 0.3	1.18 ± 0.2	1.20 ± 0.2	1.43 ± 0.2 **	1.49 ± 0.3 **	1.21 ± 0.2

* p < 0.05, ** p < 0.01 for comparisons with the corresponding control cells. # p < 0.05, ## p < 0.01 for comparisons between MF and MA. ^ p < 0.05, ^^ p < 0.01 for comparisons with CS-treated cells. ′ p < 0.05, ″ p < 0.01 for comparisons with N-treated cells.

and Figure 2 present quantitative data and representative bivariate dot plots from dual-fluorescence analyses of MF and MA stained with the green oxidative stress–sensitive probe H2DCFDA and a red fluorescent anti-TNFα monoclonal antibody detecting inflammatory activation.

Double-labeled cells were acquired on a Beckman Coulter CytoFlex flow cytometer and displayed as fluorescence density plots to characterize the relationship between oxidative stress (green channel) and the key pro-inflammatory cytokine TNFα (red channel) at the single-monocyte level. For analytical purposes, each plot was arbitrarily partitioned into four quadrants: lower left (LL, low green/low red), lower right (LR, high green/low red), upper left (UL, low green/high red) and upper right (UR, high green/high red). Quadrant boundaries were defined on untreated control cells to yield an equal distribution of 25% of events per quadrant, and these gates were subsequently applied to cells exposed to ECE 1–4, CS and N. In panel MA A, the dotted line denotes gating parameters transferred from MF, whereas the black arrow indicates the shift between MF and MA and the directionality of the concomitant, typical increase in both fluorescence signals.

ECE treatment produced different numerical and graphical patterns in adhering and non-adhering cells. Generally, ECE-treated cells were shifted upright (higher oxidative stress and higher inflammation) and stratified more clearly. This tendency was much stronger in the subpopulation of adhering cells, where a new highly active subpopulation appeared (Figure 2 MA, D. Compared to control cells, almost all ECEs altered the cell distribution in quadrants. The most spectacular changes in MF were induced by ECE 4 (srtrawberry) in the UR quadrant, where cell numbers increased from 25% to 41% (p < 0. In MA, similar effects were generated by ECE 3 (p < 0.01) and ECE 4 (p < 0.01). There was no significant difference in cell distribution, in particular ECE, regardless of cell adherence, except for the LL square, where ECE 1–3 significantly (p < 0.01) decreased cell numbers. Generally, in MF ECEs, more or less decreased cell numbers in the UL and LL regions all decreased, while in the UR and LR zones, cell numbers significantly increased. Similar changes, but much more expressed, were observed in MA, where ECE 1–4 exposure induced a marked remodeling of the bivariate distributions, resolving two discrete subsets: an L subset with lower combined fluorescence intensity and an H subset with higher combined fluorescence intensity (panel MA, D). These L and H subset groups exhibited distinct symmetry lines (Table 2), with a predominance of TNFα over the DCF signal within the highly fluorescent H population. UR L and UR H subpopulations were clearly visible and increased significantly (p < 0.01) under ECEs compared with both control and floating cells, while UR L is relatively stable, suggesting that ECEs preferentially drive cells into high-intensity TNFα/oxidative stress status. UR L symmetry line slopes remain relatively unchanged or slightly decrease, with only a modest significant reduction for CS compared with control, suggesting a limited effect on the low-intensity arm of the UR population. UR H slopes increase significantly with ECE 3–4 and CS compared with the control, consistent with steeper intensity gradients in the high-activation compartment, again pointing to a stronger activation phenotype under these conditions. The H subset also demonstrated significantly elevated CD68 expression (p < 0.01) vs L cells, consistent with an enhanced biochemical activation phenotype. The URH/URL CD68 ratio rises across ECE 1–4 versus control and CS, indicating enrichment of CD68-high cells in the most activated UR H region. CS exposure produced different patterns where significantly more cells were shifted to the UR and LR quadrants, while N generated predominantly UR shift (p < 0.01) with two clearly distinct subpopulations indicating a possible role of N in cell profiling.

4. Discussion

Monocytes and macrophages play a critical role in inflammation and oxidative stress as key components of the immune system [13]. Our main finding is that ECE exposure selectively activates small monocyte subpopulations, inducing a subclinical inflammation phenotype that is weaker and distinct from, but qualitatively similar in some aspects, to the response to CS. ECEs in whole-monocyte populations induced only modest cytotoxicity, oxidative stress, and SOD responses compared with CS, which produced clear, statistically significant increases in all three parameters. In cultured monocytes, only selected ECE flavors (notably ECE 2 and ECE 4) slightly raised oxidative stress, which corroborates our previous results [24]. SOD activity was significantly elevated only after CS exposure, supporting the view that ECEs are less toxic to monocytes than CS but not biologically inert. A key result is the marked heterogeneity of total monocyte population responses to ECEs, reflected by the threefold higher standard deviation of oxidative stress signals compared with control or N-treated cells. This dispersion suggests that only a subset of monocytes becomes strongly activated, consistent with the concept of selective activation rather than uniform stimulation of the entire population. Adherent monocytes exhibited a more activated phenotype than non-adherent cells (MF), independently of treatment, with higher CD11b, CD54, intracellular TNFα and oxidative stress, while CD11a and phagocytosis were comparable between the two fractions. This pattern matches the recognized biology of monocyte-to-macrophage transition, where adherence associates with cytoskeletal remodelling, integrin upregulation and enhanced inflammatory competence [13,25].

ECEs, to some extent, amplified this pro-adhesive and pro-inflammatory phenotype, significantly increasing the proportion of adherent cells from roughly 28% in control cultures to up to 44% for several ECEs. All four ECEs doubled the expression of CD11a, CD11b and CD54 in adherent cells, whereas CS and nicotine produced less consistent changes, with N significantly enhancing only CD11b. These data reaffirm prior observations [24,25] and indicate that ECEs, despite lower overall toxicity, efficiently prime monocytes for adhesion and possibly also for tissue retention by upregulating integrins and ICAM-1, processes directly linked to the early stages of vascular inflammation and atherogenesis.

ECE exposure increased DCF fluorescence more strongly in adherent than in floating cells, although high variability limited statistical significance in some comparisons. Published results point to the role of oxidative stress in cigarette smoke and e-cigarette effects [24,26,27,28]. CS produced the most pronounced oxidative stress (about fivefold increases in both MF and MA), which is consistent with other data [29,30,31], while N alone had a negligible effect, underscoring the role of non-N aerosol components in redox imbalance. Importantly, all ECEs significantly decreased phagocytic activity but only in MA, with up to a 50% decrease in the ECE 2 group, and CS caused an even more significant decrease, whereas N again remained largely inactive. Similar changes were already described [32,33]. Our data suggest that ECE constituents can simultaneously drive oxidative stress and biochemical but not functional activation of monocytes, decreasing their capacity to internalize particulate material and potentially to process and present antigens, which is consistent with published data [34].

The increase in TNFα was especially notable for ECE 4. Other ECEs elicited more moderate TNFα elevations, consistently more pronounced in the adherent fraction. Such pro-inflammatory effects of ECE have already been described [35,36]. Given the central role of TNFα in orchestrating inflammatory cascades and its established contribution to endothelial dysfunction, the selective but robust TNFα induction by specific e-liquid flavors (strawberry in the present flavor panel) is particularly concerning. These findings support the notion that flavoring agents may exert unanticipated pro-inflammatory effects [37,38].

Bivariate DCF-TNFα cytograms show that ECEs reshape monocyte populations beyond bulk averages by driving cells from UL/LL (double-negative or weakly positive) toward UR/LR, i.e., higher oxidative stress and/or TNFα. This effect is stronger in adherent cells, where ECE 3–4 generate a distinct high-intensity UR subset (UR H) with concomitantly elevated oxidative stress and TNFα-for example, increasing UR cells in floating monocytes from 25% to 41% under ECE 4. Within UR, ECEs mainly expand UR H while UR L remains relatively stable, indicating enrichment of strongly activated rather than mildly activated cells. UR H shows a higher UR H/UR L CD68 ratio, consistent with a macrophage-like phenotype, and steeper UR H symmetry line slopes under ECE 3, ECE 4 and CS indicate tightly coupled amplification of oxidative stress and TNFα. These changes define an ECE-driven emergence of a small (<10%) but potentially critical monocyte/macrophage subset with high oxidative burden, strong TNFα expression and increased CD68, compatible with a pro-atherogenic, tissue-injurious phenotype.

Across almost all readouts, CS produced the highest levels of oxidative stress, SOD induction, and, in many cases, the largest shifts toward UR and LR quadrants, confirming its superior toxicity relative to ECEs. However, the pattern, rather than the absolute magnitude, of activation induced by ECEs often resembled CS, particularly in terms of integrin upregulation, redistribution toward oxidative/inflammatory quadrants and emergence of UR H subsets in adherent cells. This resemblance is particularly evident for ECE 3 and ECE 4, which exerted effects comparable to CS on UR enrichment, UR H slope steepening and UR H/UR L CD68 ratios. In our previous study with alveolar epithelial cells, considerable heterogeneity in biological effects among ECE flavorings was observed [23]. N alone, used at concentrations comparable to those in ECE-conditioned media, had remarkably limited impact, with modest effects mainly restricted to CD11b expression and quadrant distribution in a few conditions. The relative quiescence of N-treated cells, juxtaposed with the strong responses elicited by complex ECE and CS aerosols, reinforces the concept that solvent matrix, degradation products (e.g., carbonyls) and flavoring chemicals, rather than N itself, are the main drivers of the inflammatory and oxidative stress responses [39,40].

The present data support the concept of ECE-induced subclinical inflammation, in which only a fraction of monocytes adopt a highly activated phenotype characterized by increased adhesion, oxidative stress, TNFα production and phagocytosis. In vivo, such selectively activated monocyte/macrophage subsets could preferentially accumulate at sites of endothelial activation, contributing to low-grade vascular inflammation, plaque initiation or progression, and exacerbation of pre-existing lung and cardiovascular disease despite the lower bulk toxicity of ECEs compared with CS. The strong modulation of responses by flavor type further suggests that the risk associated with e-cigarette use is not uniform across products and may be substantially underestimated when extrapolated from N or base-solvent exposures alone.

Several limitations need consideration. This study uses ex vivo different pulled human monocytes and a defined set of flavors at a single exposure duration, and aerosol “saturated” culture medium, which may not fully recapitulate chronic, intermittent in vivo vaping patterns or complex host–environment interactions. Flow cytometric analyses focus on TNFα, oxidative stress, selected integrins and CD68; additional markers of inflammasome activation, macrophage polarization (M1/M2), and cell death pathways would help to refine the role of mononuclear cells. Finally, the statistically significant but numerically modest shifts in some parameters, together with considerable variability, call for GC-MS analysis of ECEs, replication in individual donor cohorts and in vivo translational models.

5. Conclusions

In summary, ECE, although less globally cytotoxic than cigarette smoke, is a potent modulator of human monocyte biology, driving integrin-dependent adhesion, oxidative stress, TNFα production and phagocytic activity in a subset of cells that display a macrophage-like, CD68-high, biochemically activated phenotype. This selective activation manifests as a shift of monocytes from resting-like to oxidative/inflammatory quadrants on DCF–TNFα scatterplots, with the strongest effects observed in adherent cells and for specific flavor formulations, particularly ECE 3 and ECE 4. These findings argue against the assumption that e-cigarette use is biologically benign and instead support the view that vaping can sustain subclinical inflammation and potentially contribute to chronic cardiopulmonary risk, especially when exposure is prolonged, involves certain flavors, overlaps with CS exposure or occurs in individuals with pre-existing inflammatory or cardiovascular conditions.