Numerous studies have demonstrated the broad effects of alcohol on virtually all organ systems in the human body, including the cardiovascular system, gastrointestinal system, nervous system, and immune system (Albert et al., 1999; Belleville, 2002; Ronksley et al., 2011; de la Monte and Kril, 2014; Engen et al., 2015; Louvet and Mathurin, 2015; Pasala et al., 2015; Barr et al., 2016; Bishehsari et al., 2017; Obad et al., 2018; Le Dare et al., 2019). Interestingly, the impact of alcohol on human health and mortality appears to be dose-dependent (Barr et al., 2016). Light to moderate alcohol consumption is associated with beneficial effects on cardiovascular and autoimmune diseases (e.g., rheumatoid arthritis or hyperthyroidism) (Maxwell et al., 2010; Gil-Bernabe et al., 2011; Carlé et al., 2012; Carlé et al., 2013; Nurmi et al., 2013). On the other hand, high-dosed and chronic consumption has been linked to cardiomyopathies, increased risk of viral or bacterial infections, and a variety of cancers (e.g., pharynx, larynx, esophagus, liver) (Happel and Nelson, 2005; Baliunas et al., 2010; Li et al., 2014; Turati et al., 2014; Le Dare et al., 2019; Fernández-Solà, 2020). However, the observed effects of alcohol on the human body are complex and a topic of controversial discussion in the scientific community. Several recent studies have found growing evidence that even moderate alcohol consumption increases health risks, leading to the suggestion that the beneficial effects of alcohol depend on sex, age, background diseases, and region (Topiwala et al., 2017; Bryazka et al., 2022; Topiwala et al., 2022).

Binge drinking (i.e., high-dosage alcohol intoxication) is a harmful drinking pattern defined as alcohol consumption that elevates the blood alcohol concentration (BAC) to 0.8‰, which typically occurs after five or more drinks by men and four or more drinks by women within 2 h (National Institute on Alcohol Abuse and Alcoholism, 2004; Molina and Nelson, 2018). It leads to increased mortality and is mainly popular among young adults but also occurs in high frequency among elderly adults (Courtney and Polich, 2009; Han et al., 2019; Surial et al., 2021). Consuming large amounts of alcohol in a short period of time results in high BAC which is concerning due to several detrimental pathophysiological mechanisms that have been identified. Alcohol can permeate all organ tissues causing inflammation, oxidative stress, mitochondrial injury, and cell membrane perturbation (Molina and Nelson, 2018). Furthermore, several studies have underscored the profound impact of ethanol on the gene expression of pattern recognition receptors (PRR), activation states, cytokine expression, phagocytic activity, reactive oxygen species (ROS) release, cell counts, and migration in both innate and adaptive immune cells (Schmidt and Lint, 1972; Saitz et al., 1997; Molina et al., 2003; Molina et al., 2014; Molina and Nelson, 2018; Stadlbauer et al., 2019).

The examination of cells’ biophysical properties has attracted growing attention in recent research, as new and more feasible methods were established for cell analyzing (Gossett et al., 2012; Byun et al., 2013; Otto et al., 2015; Guillou et al., 2016; Nyberg et al., 2017). The substantial role of the cytoskeleton in essential cell functions like activation, mitosis, migration, and phagocytosis provides the opportunity to use cell mechanical characteristics as promising biomarkers for biological processes in health and disease (Di Carlo, 2012). Until recently, it was challenging to measure more than a few or several hundred cells per hour with methods like atomic force microscopy (AFM), micropipette aspiration, or optical stretcher (Binnig et al., 1986; Hochmuth, 2000; Guck et al., 2001). These techniques did not provide the means for analyzing large cohorts of cell populations in screening procedures or routine clinical usage. For this reason, among others, cell mechanical measurements took only place in basic biophysics research (Di Carlo, 2012).

While numerous studies have investigated the effect of alcohol on the cellular mechanisms of leukocytes, the impact on their mechanical properties remains largely unexplored. Recognizing ethanol-specific changes in cell mechanics could illuminate alterations in the immune constitution of individuals with acute alcohol intoxication or those diagnosed with alcohol use disorder (AUD), potentially marking these alterations as biomarkers. In response to this knowledge gap, our pilot study was designed to employ real-time deformability cytometry (RT-DC)—a novel tool for high-throughput cell mechanical analysis (Otto et al., 2015). Specifically, we examined the potential effects of acute alcohol exposure on the cell mechanics of both steady-state leukocytes ex vivo and activated leukocytes in vitro.

Clinical and animal studies have revealed that alcohol consumption, particularly at acute or chronically high levels, not only increases the susceptibility to infections but also contributes to poorer outcomes and slower recovery in traumatic injuries such as burns or bone fractures (Schmidt and Lint, 1972; Saitz et al., 1997; Silver et al., 2008; Sears et al., 2011; Lauing et al., 2012; Shults et al., 2015; Shults et al., 2016). This is primarily attributed to alterations in critical immune cell functions in both innate and adaptive immunity.

Recent research has focused on the morphological and mechanical characteristics of cells as potential biomarkers for various diseases. It is hypothesized that specific disease-related alterations in cells’ biophysical properties could be detected using methods such as RT-DC (Guck and Chilvers, 2013; Otto et al., 2015). Up to now, a few approaches have been made by characterizing the blood cells in patients with viral respiratory tract and Epstein-Barr-virus (EBV) infections (Toepfner et al., 2018), COVID-19 patients (Kubánková et al., 2021) and recently, singularized suspended cells from the human colon for differentiating between healthy and tumorous tissues (Soteriou et al., 2023). Interestingly, the distinct leukocyte subsets showed different cell mechanical responses, depending on the viral pathogen involved (Kubánková et al., 2021). These alterations primarily involved changes in cell size and deformability. The successful identification and validation of specific cell mechanical profiles could pave the way for incorporating these parameters into diagnostic applications or as biomarkers.

Ethanol, a small amphiphilic molecule, interacts with biological membranes in various ways. It predominantly localizes within the headgroup region of the lipids, where it can form hydrogen bonds with the phosphate and carbonyl groups (Rottenberg, 1992; Feller et al., 2002; Stetter and Hugel, 2013). This interaction contributes to altered membrane structures, potentially leading to observed effects on membrane properties, such as an increase in permeability, fluidity, and elasticity (Chin and Goldstein, 1977; Komatsu and Okada, 1997; Ly and Longo, 2004). Furthermore, ethanol can interact with membrane-associated proteins such as ion channels, receptors, adhesion molecules and enzymes, causing structural changes, and influencing vital cell functions (Escriba et al., 2008; Tóth et al., 2014). Additionally, evidence suggests that the cytoskeleton—a complex network of microtubules, actin, and intermediate filaments—may also be vulnerable to ethanol toxicity (Yoon et al., 1998; Tomás et al., 2003; Tomás et al., 2005; Fletcher and Mullins, 2010; Tóth et al., 2014; Adhikari et al., 2023). However, most of these findings are derived from either animal or membrane model studies and their comparability to in vivo or ex vivo studies might be limited. Our findings demonstrate that a single episode of binge drinking does not impact the biophysical cell properties of ex vivo analyzed steady-state lymphocytes, monocytes, or neutrophils within the first 2 h post-binge drinking. It is important to note that this study, conducted using RT-DC, focused on deformability, Young’s modulus, cell size, and volume, while membrane fluidity and permeability were not investigated. Consequently, further research is needed to explore these biophysical parameters that have shown alterations in previous studies.

In line with prior studies, immunophenotyping results revealed significantly altered relative cell counts of leukocyte subtypes. Several studies demonstrated a disbalance between naive and memory CD4 and CD8 T cells, resulting in a shift toward a memory phenotype (Cook et al., 1994; Cook et al., 1995; Song et al., 2002). Higher numbers of memory T cells have been linked to chronic inflammatory disorders, autoimmunity and pathologies that primarily occur in older individuals (Hakim and Gress, 2007; Chou and Effros, 2013). Further, it is suggested that the observed shift in T cell phenotype in patients with alcohol use disorder (AUD) is analogous to that observed in immunosenescence (Zuluaga et al., 2020). Additionally, we observed a significant decrease in relative NK cell counts, which has been described for early patients with AUD (Perney et al., 2003). Conversely, patients with alcohol-related cirrhosis seem to have an increased NK cell percentage with elevated cytotoxicity, compared to healthy individuals (Sehgal et al., 2020). Such discrepancies might be a result of different stages of liver disease highlighting the time and dose dependency of alcohol-induced alterations in the immune system (Zuluaga et al., 2022). Consistent with prior research, we did not observe significant alterations in B cell subtypes, monocytes, or granulocytes within the first 2 h after binge drinking (Afshar et al., 2015; Haag et al., 2022).

Our in vitro studies yielded results similar to the ex vivo measurements with steady-state leukocytes. Apart from a change in the SD cell area of lymphocytes, no significant alterations in biophysical parameters were observed in leukocytes co-incubated with 26.04 mmol/l (1.2‰) ethanol. In our investigation, we noticed a significant increase in neutrophil cell deformability and the SD cell area, alongside a decrease in neutrophils and monocytes Young’s modulus, indicating an overall reduction in cell stiffness. Previous research, utilizing a membrane model, deduced that the localization of ethanol molecules in the headgroup area of membranes leads to an expansion of the headgroup area, causing an overall thinning of the membrane. This enlarged area diminishes the interactions between these headgroups, which subsequently leads to a reduced Young’s modulus (Stetter and Hugel, 2013). While these observations were conducted in considerably higher ethanol concentrations, ranging between 10% and 40%, our findings propose that such effects might be discernible even at lower concentrations.

In our study, we aimed to learn about ethanol’s influence on the biophysical cell properties of leukocytes during the stimulation process. LPS is detected by PRR and prompts an innate immune response via the toll-like receptor (TLR)-4 complex, especially in monocytes and neutrophils (Ciesielska et al., 2021). As anticipated, we observed no significant alterations in the biophysical properties of the lymphocyte population, attributable to their lack of responsiveness to short-time LPS exposure (Chaiwut and Kasinrerk, 2022). Interestingly, LPS stimulation led to a significant change in SD cell area of lymphocytes, implicating a heterogenous response to LPS in the lymphocyte population. Notably, NK cells, which are known to respond to LPS are also classified within the lymphocyte population in RT-DC measurements and may cause this observation (Kanevskiy et al., 2013; Otto et al., 2015). Consistent with prior research, monocytes exhibited significantly increased deformability upon LPS stimulation and decreased compressibility, as evidenced by an increase in Young’s modulus (Ravetto et al., 2014). The observed effects in monocytes after LPS exposure may indicate two potential processes: cytoskeletal reorganization for migration and differentiation into macrophages or dendritic cells (Vicente-Manzanares and Sánchez-Madrid, 2004; Jakubzick et al., 2017). Indeed, studies have noted alterations in elastic properties during cell differentiation, specifically an increase in cell stiffness (Darling et al., 2008; Yu et al., 2010). Remarkably, higher deformability was not evident when cells were co-incubated in ethanol. In vitro studies demonstrated that exposure of human monocytes to 25 mmol/l ethanol reduces the production of TLR 4-mediated proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha in response to LPS, while it increases the anti-inflammatory cytokine IL-10 (Szabo et al., 1996). Additionally, it initiates a signal cascade, involving the heat shock transcription factor (HSF)-1, leading to the downregulation of TLR-4 (Mandrekar et al., 2008). It is well established that internalization and downregulation of TLR 4 effectively terminate the pro-inflammatory immune reaction (Ciesielska et al., 2021). We hypothesize that the previously described anti-inflammatory effects of ethanol, and in turn the absence of a pro-inflammatory response, impede the reorganization of the cytoskeleton. The underlying mechanisms might be pleiotropic, considering the variability in membrane constitution among different subpopulations and the reported effects of ethanol on the cell membrane proteins, the bilayer itself, transcription factors, and the cytoskeleton (Tóth et al., 2014; Poggi et al., 2015).

LPS stimulation also resulted in a significant increase in neutrophil deformability, cell area and volume. Unlike monocytes, these responses were not hampered by co-incubation with ethanol, underscoring the heterogenous response of immune cells to ethanol. Recent research observed enhanced priming and ROS production in isolated neutrophils following binge alcohol consumption in healthy volunteers, while others report the opposite effects (Stadlbauer et al., 2019; Haag et al., 2022). Notably, both studies found an increase in non-phagocytosing cells. In our study, the stimulated as well as co-incubated cells demonstrated similar significant changes compared to the control group. Hence, further research is required to investigate a potential correlation between the phagocytosis capacity and biophysical parameters and whether these can be determined via RT-DC.

In this study, we observed a significant increase in lymphocyte cell deformability and a decrease in cell stiffness during stimulation with CytoStim™. As APCs, such as monocytes, are critical for T cell stimulation with CytoStim™, we hypothesize that the observed increase in monocyte deformability indicates their involvement in this process. Moreover, there is evidence suggesting that IFN-gamma, produced by Th cells 1, NK cells, and CD8 T cells, can independently activate monocytes and polarize them toward a highly pro-inflammatory macrophage phenotype, possibly resulting in the observed altered biophysical properties (Luque-Martin et al., 2021). In contrast to the changes observed during lymphocyte activation, monocytes did not display increased deformability when co-incubated with ethanol. This observation gains importance considering that in vitro studies have reported a disruption in the antigen presentation abilities of monocytes when exposed to ethanol, resulting in inhibited superantigen-induced T cell proliferation (Szabo et al., 1993; Szabo et al., 1995). Interestingly, ethanol co-incubation did not hamper the increase in lymphocyte deformability, raising the question of whether impaired antigen presentation by monocytes and lymphocyte proliferation might be detectable via RT-DC at later time points in the stimulation process. In contrast to the stimulation with LPS, no similar increase in cell size was observed in monocytes when stimulated with CytoStim™. This intriguing discrepancy may be related to an apoptotic response triggered in monocytes by CytoStim™. Past research has demonstrated that the superantigen staphylococcal enterotoxin B (SEB) induces apoptosis in monocytes (Takahashi et al., 2001). Interestingly, this apoptotic response was inhibited when LPS was present (Takahashi et al., 2001). As CytoStim™ operates similarly to SEB, it might also induce apoptosis and thereby prevent the increase in cell size that we observed with LPS stimulation.

While our study provides meaningful insights into the impact of ethanol on the biophysical properties of immune cells, it is important to acknowledge several limitations associated with this being a pilot study. One significant limitation is the small sample size which reduces the statistical power and generalizability of our results. Another notable limitation is the lack of a follow-up phase; the potential effects of alcohol intoxication on cell mechanical properties might be detectable at later time points, beyond those measured in our study. A follow-up phase would have also allowed us to monitor the duration of the observed changes in immune cell counts and to determine when the phenotype reverts to its normal state. For instance, the effects were transient in certain studies and faded within hours (Stadlbauer et al., 2019; Haag et al., 2022). Our study design did not account for these time-dependent changes. This calls for future studies to incorporate time-course analysis with a higher sample size for a more comprehensive understanding of cellular kinetics post-ethanol exposure. Our examination primarily focused on cellular populations that are heterogeneous in nature, encompassing multiple cell types with contrasting functions within the immune system. This heterogeneity necessitates a more detailed characterization of each individual cell population. For instance, the significance of RT-DC measurements could be enhanced by applying the recently developed fluorescence module, allowing better discrimination between different cell types while characterizing the cell mechanical properties (Rosendahl et al., 2018). Furthermore, we did not perform immunophenotyping on the samples from our in vitro experiments. Future studies could contrast ex vivo and in vitro immunophenotyping results to investigate whether the observed effects are directly induced by ethanol or if there are additional in vivo factors at play. Given the scope of this pilot study, we did not examine the expression of activation markers, cytokines, or changes in actin polymerization, which limits our understanding of the functional implications of the observed biophysical changes.

In conclusion, our study suggests that a single episode of binge drinking does not significantly affect the biophysical properties of steady-state leukocytes in healthy individuals. However, it does lead to significant changes in the relative cell counts and a shift toward a memory T cell phenotype. On the other hand, when cells were stimulated, monocytes showed a susceptibility to ethanol, manifested as a hindered increase in cell deformability. This indicates that ethanol might inhibit the reorganization of the cytoskeleton during the stimulation process. Additional studies are necessary to confirm our observations, further assess the presence of ethanol-specific cell mechanical profiles in both acute and chronic exposure, and explore the potential of biophysical properties as biomarkers or prognostic indicators in alcohol-related health damage.