The immune system is influenced by a myriad of environmental factors, including alcohol consumption. In 2019, 69% of U.S. adults aged 26 years or older reported alcohol use in the past month (1). Importantly, social stresses associated with the COVID-19 pandemic have led to increased sales of alcoholic beverages: sales were 3.6% higher in March 2020 and 15.5% higher in April 2021 when compared to a 3-year average from the same month in 2017-2019 (2). Further, alcohol-related deaths have been rising at an astonishingly quick pace in recent years: fatalities due to alcohol rose by 25.9% between 2019 and 2020, compared to a 16.6% increase in age-adjusted mortality rates from all causes (3). Based on this rapidly escalating prevalence of alcohol consumption, we will likely continue to see a rise alcohol-associated morbidity and mortality well into the future.

Alcohol’s effects on the immune system vary based on the amount and duration of consumption. Broadly speaking, alcohol intake can be classified as “moderate” or “excessive” (4). The U.S. Centers for Disease Control and Prevention describe “moderate” alcohol intake as 1-2 drinks per day for males or 1 drink per day for a female (4). “Excessive” alcohol consumption includes binge drinking, or that which brings blood alcohol concentration (BAC) above the “legal limit” for driving (80 mg/dL; usually 5+ drinks for a male or 4+ drinks for a female within 2 hours) and heavy drinking, considered 14+ drinks per week for a male or 7+ drinks for a female (5). In addition, human studies characterize “alcohol use disorder” (AUD) as the inability to modify or discontinue alcohol use despite adverse consequences to one’s personal or work life, and is clinically measured by several social and psychological parameters (6).

Epidemiological studies have shown that moderate alcohol intake is generally associated with protective health effects, such as decreased risk of cardiovascular disease [reviewed in (7)], lower concentrations of circulating inflammatory biomarkers (8), and lower risk of all-cause mortality in humans (9). In contrast, other studies have found an increased risk for all-cause mortality in adults with heavy alcohol consumption [> 14 and > 7 drinks per week for men and women, respectively (9), or 5+ drinks on a single occasion at least once per week in the past year (10)]. Additionally, chronic alcohol users often have health complications associated with alcoholic hepatitis and its treatment, such as invasive aspergillosis (11) and infections of the lower respiratory and urinary tracts (12). Mouse models have similarly shown that excessive ethanol exposure is linked to deleterious effects on immunity; these include decreased responsiveness to Toll-like receptor (TLR) 2, 4, and 9 agonists (13), diminished phagocytic capacity (14, 15), impaired respiratory function (16), and increased airway neutrophils following acute lipopolysaccharide-induced lung injury (17). Neutrophil function is also compromised due to excessive ethanol consumption. For example, chronic ethanol exposure is associated with impaired neutrophil chemotaxis to the airways following pulmonary Aspergillus fumigatus infection, along with attenuated phagocytosis, fungal killing, and reactive oxygen species production in A. fumigatus-challenged neutrophils from ethanol-fed mice (18). Others have demonstrated that acute ethanol treatment (6 g/kg) decreases neutrophil infiltration into the peritoneal cavity following cecal ligation and puncture, accompanied by diminished production of neutrophil extracellular traps and bacterial killing (19).

Excessive alcohol use has been linked to increased susceptibility to infectious diseases such as pneumonia, with the most common bacterial cause being Streptococcus pneumoniae (20). The estimated annual incidence of pneumonia in the United States is 24.8 cases per 10,000 adults (21), and approximately 3.5% of pneumonia patients had an AUD at diagnosis (22). In fact, meta-analyses have found a dose-dependent, linear relationship between alcohol consumption and relative risk for contracting pneumonia (23, 24). In addition, patients with alcohol use disorder are more likely to have severe invasive disease that requires hospitalization (25) and adults who drink excessively (> 60 grams per day) are 4 times more likely to die within 30 days of infection (26).

Innate immune recognition of S. pneumoniae by tissue-resident alveolar macrophages or epithelial cells stimulates the production of pro-inflammatory C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2, which recruit and activate cells, such as monocytes, macrophages, and neutrophils to the site of infection (27–29). Additionally, in the context of pulmonary injury, CXCL12 promotes neutrophil migration and retention in the lung (30, 31). Important chemokines for the production and mobilization of granulocytes from the bone marrow are granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Following an inflammatory stimuli such as infection, G-CSF and GM-CSF are rapidly produced and secreted by monocytes, macrophages, fibroblasts, and endothelial cells and this corresponds with an increase in neutrophil differentiation and production in the bone marrow (32–34). Indeed, mice lacking G-CSF or its receptor have reduced neutrophil counts in the blood and bronchoalveolar lavage (BAL) fluid following Pseudomonas aeruginosa lung infection (35). While the importance of innate immune cells in the response to S. pneumoniae has long been appreciated (36–38), excessive accumulation of these cells can be deleterious to host tissue if the inflammatory response is not properly controlled. For example, increased neutrophil recruitment to the lungs following influenza-induced pneumonia in mice is associated with alveolar damage, pulmonary edema, and development of a phenotype similar to that seen in critically ill humans with acute respiratory distress syndrome (39).

Our current knowledge regarding the effects of acute alcohol intoxication on lung immunity is largely based on animal studies using supra-physiologic doses of alcohol (3-5 g/kg) (40, 41), sometimes high enough to raise blood alcohol concentration to 350-550 mg/dL (42, 43). Here, we sought to determine the effect of a 3 day lower-dose alcohol regimen (1.5 g/kg; target BAC of ~80 mg/dL) on the pulmonary response to a clinically relevant intranasal S. pneumoniae infection (44).

Excessive alcohol consumption weakens the ability of the immune system to effectively respond to pathogens (52). Here, we describe those 3 consecutive days of a more moderate ethanol exposure than what is typically used in the literature (Figures 1A, B) can significantly alter the early pulmonary response to Streptococcus pneumoniae. Although the peak BAC in our studies is approximately 80 mg/dL, considered the “legal limit” for driving in humans, it is likely that the BAC levels for intoxication in mice are comparably lower due to increased metabolism of ethanol in rodents compared to humans (53). Nonetheless, we found that ethanol exposure increases bacterial burden in the lung and decreases respiratory function within 24 hours after S. pneumoniae infection (Figures 1C, 2). Our results showing increased bacterial burden in the lungs of ethanol-exposed mice (Figure 1C) is in line with previous studies utilizing pathogens such as S. pneumoniae (54), Klebsiella pneumoniae (55, 56), and Escherichia coli (57). However, the animal models in these studies use supra-physiological levels of acute ethanol (often enough to raise BAC well above 350 mg/dL) or binge-on-chronic ethanol feeding [Lieber-DeCarli diet (58) for a total of 10 days with a 4 g/kg ethanol gavage on days 5 and 10 (55, 56)] before infection, making it challenging to directly compare results. Even so, our work expands upon previous findings by showing a similar response of increased bacterial burden in animals exposed to a much lower and shorter three-day ethanol exposure regimen (1.5 g/kg) and infected with a lower S. pneumoniae inoculum (10⁴ CFU). We believe that our model better recapitulates how humans who drink alcohol may acquire bacterial pneumonia via respiratory droplets, as our mice are given an oral gavage of ethanol at a more moderate level and then infected intranasally. Furthermore, humans tend to drink alcohol for social motives (59) putting them in close proximity to others who may be infected, or more commonly, those who are asymptomatic carriers of S. pneumoniae (60).

Previous studies have found that respiratory disease is associated with impaired lung function using whole body plethysmography, including pneumococcal (61) and SARS-CoV2 infection (62), and bleomycin-induced lung injury (63). Further, our group has shown that multi-day ethanol exposure leads to respiratory dysfunction following traumatic injury, such as a cutaneous scald (16, 64). The increased penh values suggest altered airflow patterns in response to airway inflammation (65, 66), while the higher expiratory time and corresponding decreased Rpef values suggest airway narrowing/obstruction (67, 68) and airway collapse with increased airflow resistance (69, 70) in ethanol- compared to vehicle-exposed infected mice. Increased leukocyte accumulation, pulmonary edema, and alveolar wall thickening observed histologically (Figure 5A; Supplementary Figure 2) likely contribute to the impaired respiratory function observed by plethysmography. One caveat to our lung function data is that we observed mortality during the studies so respiratory parameters are representative of surviving animals only. On day 4, 6% and 14% of the vehicle- and ethanol-exposed infected animals, respectively, died. An additional 12% and 8%, respectively, of the remaining animals died on day 5; we did not observe any further mortality through day 7 post-infection (data not shown). Nevertheless, our study is the first to our knowledge that demonstrates an effect of multi-day lower-dose ethanol exposure on respiratory parameters following S. pneumoniae infection.

Neutrophils are critical early responders to respiratory pathogens, but their presence and activity at infection sites can be detrimental if not properly controlled. Using several different pathogens, animal studies have demonstrated that the host is more susceptible to severe pneumonia and death when neutrophils are depleted or otherwise unable to reach the lung (36, 71), but also when excessive neutrophil accumulation and their ineffective clearance leads to tissue damage (72–75). Our results show that ethanol-exposed mice had a higher pulmonary bacterial burden at 24 hours after infection (Figure 1C) despite increased numbers of pulmonary neutrophils (Figure 3B) and correspondingly higher expression of chemokines involved in neutrophil recruitment, such as Cxcl1 and Cxcl2 (Figure 4A). Likewise, the upregulation of Csf3 in the lungs and increased levels of G-CSF in the serum of ethanol-exposed infected mice (Figures 4B, C) further suggests an immune response geared toward granulopoiesis and neutrophil homing to the lung. Indeed, others have shown that G-CSF mRNA levels are markedly increased in lung tissue from animals challenged intratracheally with E. coli but did not observe appreciable levels of G-CSF mRNA in spleen, liver, or kidney tissue from the same animals (76). This suggests that the lung is a primary source of G-CSF production early after pulmonary infection.

Previous work from our lab has shown that prior ethanol exposure leads to excessive accumulation of neutrophils (77, 78) and apoptotic cells (78) in the lungs of mice up to 24 hours following burn injury, compared to vehicle-treated injured mice. Although not directly tested in these studies, others have shown that ethanol exposure decreases phagocytic capacity in both alveolar macrophages (14, 15, 79–81) and neutrophils (82–84), along with decreased efferocytosis by alveolar macrophages (17). Our results showing increased bacterial burden in the lung despite higher numbers of neutrophils may suggest that either the tissue-resident alveolar macrophages or the infiltrating neutrophils are less efficient at properly phagocytosing and/or breaking down S. pneumoniae within the first 24 hours after infection. Our IHC staining of infected lung tissue would suggest the former (Figure 6), although we cannot rule out that the recruited neutrophils are also functionally impaired. Additionally, the presence of Ly6G-negative airway cells could indicate apoptotic macrophages that were not detected by flow cytometry. It is also possible that ethanol treatment in our mice indirectly alters neutrophil apoptosis due to the hyper-inflammatory lung microenvironment following infection (85) and/or impairs efferocytosis of apoptotic neutrophils by alveolar macrophages or infiltrating macrophages; this question merits further evaluation and clarification. Indeed, others have reported delayed neutrophil apoptosis after acute ethanol exposure followed by a “second hit” to the immune system (86, 87). It has been shown that blood neutrophils from ethanol-exposed burned animals had decreased expression of pro-apoptotic proteins such as caspase-3 and Bax, and correspondingly decreased apoptosis as measured by histone-associated DNA fragments (86). Further, delayed neutrophil death appears to be a common characteristic of human inflammatory lung diseases such as cystic fibrosis, pneumonia, and idiopathic fibrosis, as well as in cancer with associated neutrophilia (87). Importantly, in our model, if recruited pulmonary neutrophils are not able to properly die via apoptosis and be cleared by alveolar macrophages, they may undergo secondary necrosis, releasing toxic mediators such as elastase and reactive oxygen species, and inducing lung damage in our ethanol-exposed animals (88, 89).

Inflammation is a necessary process to restore homeostasis after infection. Resident immune cells in the upper and lower respiratory tract detect pathogen associated molecular patterns on invading microorganisms and initiate a signaling cascade leading to mobilization and migration of leukocytes to the site of infection. The lungs of our infected animals showed an early accumulation of peri-vascular and airway leukocytes in animals with prior ethanol exposure (Figures 4A, B). This suggests that recruited immune cells are able to reach the lungs but appear to be less efficient at clearance of S. pneumoniae as noted by CFU counts at 24 hours post-infection in our ethanol-exposed mice (Figure 1C). While we failed to see differences in the number of alveolar or infiltrating macrophages in lung homogenates by flow cytometry (Figure 3), it is possible that ethanol exposure impairs monocyte trafficking to the lung, as others have previously shown (90).

Immunohistochemistry staining of lung sections provided valuable insight into the effect of ethanol on neutrophil and bacterial antigen localization, as well as bacterial internalization, following S. pneumoniae infection. We did not observe any gross difference in neutrophil proximity to S. pneumoniae, suggesting that neutrophils are able to migrate toward the infection site at 24 hours post-infection in our model. However, it is possible that neutrophil chemotaxis is impaired at earlier or later time points post-infection, or in other areas of the lung. Further, there was no significant difference in neutrophil or bacterial antigen staining intensity (Supplementary Figure 1), however, we are careful not to rely solely on this result since the quantification is from a single tissue section from each animal. Additionally, the S. pneumoniae antibody used for IHC is a whole cell serotype blend and will therefore bind to viable and non-viable bacteria and also closely related bacterial species; therefore, quantification of S. pneumoniae antigen by IHC may not directly relate to CFU counts of viable bacteria from lung homogenates.

The results presented here require further clarification in future studies. A strength of this work is the demonstration of increased neutrophils and S. pneumoniae in whole lung homogenates of ethanol-exposed infected animals at 24 hours, and visualization of the localization of each within the lung. In future experiments, we will characterize the number and functional capacity of neutrophil and macrophage populations in the airways following infection by isolation of these cells from BAL fluid (91). Advanced flow cytometry analysis of blood, BAL fluid, and whole lung homogenates will identify the changing inflammatory cell populations and discriminate cell death (apoptosis or necrosis) in each. Additionally, it would be useful to determine which subset of pulmonary cells are expressing Cxcl1, Cxcl2 and Csf3, as this may yield a focused therapeutic target to improve health outcomes in alcohol consumers with pneumococcal pneumonia. Finally, because S. pneumoniae was still present in the lungs at 24 hours post-infection, it is imperative to examine the later innate and subsequent adaptive immune response in our model and determine whether complete resolution of the infection is altered due to ethanol exposure.

In summary, we show here that ethanol exposure—at a dose relevant to human consumption—results in higher lung bacterial burden despite an increased presence of pulmonary neutrophils following intranasal S. pneumoniae infection. An accumulation of leukocytes was visualized in the airways and peri-vascular space, and likely contributes to the respiratory dysfunction seen in our infected animals. Additionally, we noted differences in S. pneumoniae internalization in macrophages from our ethanol-exposed infected animals, which likely contributes to the increased inflammation noted in our mice and could lead to delayed resolution of the infection. Taken together, these findings contribute to our knowledge of how short-term ethanol consumption at physiological doses can alter pulmonary immunity to respiratory infection. Future studies aimed at understanding the mechanisms underlying this exacerbated neutrophilic response could improve health outcomes in pneumonia patients who drink alcohol.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by IACUC University of Colorado Anschutz Medical Campus.

HH and KN designed and carried out the experiments. HH analyzed the data and wrote the manuscript. RM provided guidance on study design, and analyzing and interpreting flow cytometry data. DB provided critical input on experimental strategy and panel design for the flow cytometry experiments. DO blindly scored the H&E sections and provided interpretation of the lung histology. EK supervised the project. All authors provided feedback on results and critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported in part by NIH F31 AA027687 (HH), R21 AA026295 (EK), R01 AG018859 (EK), R35 GM131831 (EK), and VA 1 I01 BX004335 (EK).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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