A single alcohol binge impacts on neutrophil function without changes in gut barrier function and gut microbiome composition in healthy volunteers

Alcohol binge drinking is a dangerous drinking habit, associated with neurological problems and inflammation. The impact of a single alcohol binge on innate immunity, gut barrier and gut microbiome was studied. In this cohort study 15 healthy volunteers received 2 ml vodka 40% v/v ethanol/kg body weight. Neutrophil function was studied by flow cytometry; markers of gut permeability and inflammation (lactulose/mannitol/sucrose test, zonulin, calprotectin, diamino-oxidase) were studied with NMR spectroscopy and enzyme-linked immunosorbent assay in urine, stool and serum respectively. Bacterial products in serum were quantified using different reporter cell lines. Gut microbiome composition was studied by 16S rDNA sequencing and bioinformatics analysis. After a single alcohol binge, neutrophils were transiently primed and the response to E.coli stimulation with reactive oxygen species (ROS) production was transiently increased, on the other hand the percentage of neutrophils that did not perform phagocytosis increased. No changes in gut permeability, inflammatory biomarker, bacterial translocation and microbiome composition could be detected up to 4 hours after a single alcohol binge or on the next day. A single alcohol binge in young, healthy volunteers transiently impacts on neutrophil function. Although the exact biological consequence of this finding is not clear yet, we believe that this strengthens the importance to avoid any alcohol binge drinking, even in young, otherwise healthy persons.

Alcohol binge drinking, defined as 5 or more drinks for men and 4 or more drinks for women at one time, is the most frequent form of alcohol consumption worldwide, especially in younger people. [1] This drinking pattern is popular and leads to increased mortality and morbidity. Therefore binge drinking is a major public health issue. The behavioural and neurological consequences of binge drinking are well characterized [2][3][4].
Less is known about the systemic effects on the gut as the first organ in contact with alcohol and on the consequences on inflammation and immune function. Chronic alcohol intake can lead to increased gut permeability, bacterial translocation and alterations in the gut microbiome in animal models. [5][6][7] Recently bacterial translocation has been shown in healthy volunteers after a single alcohol binge. [8] On immune cells, acute alcohol intake seems to have dichotomous effects. On the one hand alcohol induced liver injury is driven by pro-inflammatory reactions, whereas in the long-term, immunosuppressive and anti-inflammatory effects have been described. [9] These immune effects seem to be driven by endotoxin or other bacterial products via Toll-like receptors that are translocated to the circulation via a defective gut barrier. [10] 2

Hypothesis
We hypothesize that binge drinking impairs gut barrier, increases bacterial translocation and causes inflammation. This leads to an early increase in neutrophil and monocyte function but also an increase in oxidative stress. (see Figure 1). Figure 1: Hypothesis of how alcohol consumption impacts on gut permeability and liver/phagocyte function.

Preliminary data
In previous research projects we gathered preliminary data that draw a crude picture of the influence that alcohol has on the innate immune system. In 55 abstinent patients with alcohol-induced liver cirrhosis we found that neutrophil function depends on liver synthesis. Patients with compensated cirrhosis (Child-Pugh grade A) showed a significant decrease in phagocytic activity (percentage of phagocytizing neutrophils) but a slight increase in phagocytic capacity (semi quantitative measurement of the number of engulfed bacteria). It is possible that with the increase in inactive cells the remaining functioning cells compensate this loss by increasing their capacity. In decompensated cirrhosis (Child-Pugh grade B/C) this compensation seems to be lost and neutrophil dysfunction becomes apparent.
To clarify the role of liver damage in alcohol induced immune disorder we utilized the Lieber-DeCarli model of alcohol feeding on C57BL/6 mice that exhibit similar damage to the gut barrier and microbiome as humans do but only develop very mild alcoholic liver disease (ALD). We found a significantly increased ex vivo clearance capacity of whole blood, a trend to improved neutrophil function, as well as a significantly faster, more efficient clearance of pathogens from the blood in early stages of experimentally induced sepsis. When the livers of these mice were damaged with 3,5diethoxycarbonyl-1,4-dihydrocollidine (DDC) before the alcohol feeding period, the before mentioned advantages in ex vivo function tests were lost.
We therefore, hypothesize that alcohol by itself has immune activating properties as long as the liver is still functional. However, once the liver has been damaged the effectiveness of the immune system decreases with the degree of liver disease. Data presented as mean and SD, *significant on 5% level (or significant after Bonferroni correction for multiple comparison)

Study
This study should test whether one episode of binge drinking in otherwise healthy volunteers causes bacterial translocation and innate immune activation.

Recruitment
Healthy volunteers will be recruited from the volunteer database of the Clinical Volunteers will undergo 1 screening visit and 2 study visits.
The screening visit duration is approximately 1 hour, the first study visits will last at least 5 hours with an optional observation/recovery period thereafter until the alcohol breath test is below 0.5 ‰. On the next day patients will have to perform the gut permeability test at home and will deliver the urine sample together with a stool sample and a questionnaire to the trial centre. Table 1 Schedule of assessments

Screening visit 1
Male and female healthy volunteers who accomplish the inclusion criteria and none of the exclusion criteria are eligible for the study and will undergo the following investigation procedures in the screening visit, after signing the informed consent: • Subjects will be informed about the nature of the study, including the intake of alcohol. The patients have to sign that they will not use a car or a machine on the day of the study. They have to state how they will travel home after the study (either taxi, public transport or being picked up by an adult friend or family member).
As part of this visit the following procedures will be performed: • Body weight • Vital signs: resting pulse and blood pressure

• Physical examination
• Pregnancy test in women of childbearing age • Alcohol breath test • Blood sampling via a venous access line before alcohol consumption and every hour for 4 hours.
• Consumption of 2 ml vodka 40% v/v ethanol/kg body weight in a total volume of 300 ml orange or strawberry juice (alcohol group) or consumption of 2 ml tap water/kg body weight in a total volume of 300 ml orange or strawberry juice (control group), or 75g of fructose in 300ml of water, or a mixed caloric drink (Fortimel compact, 10kcal/kg) Subjects from the alcohol group will be monitored at the Clinical Research Center until the alcohol breath test is below 0.5 ‰. Subjects will be provided with a taxi voucher.

Visit 3
24h after visit 1 they patients will have to perform the gut permeability urine test for 5h at home, collect a stool sample and fill in the self-administered hangover questionnaire and will have to bring the urine sample, the stool sample and the questionnaire to the trial site.

Sample size
In order to determine the magnitude of effect on gut permeability and innate immune function by a single alcohol binge, we intend to perform a pilot study on 46 healthy volunteers (16 in the alcohol group, 30 in the control groups).

Tests
The assays described in this section are the current state of the art. It is possible that the methodology changes until analysis or that assays need to be exchanged for technical reasons.

Gut permeability and inflammation
A ready-to-use solid-phase sandwich ELISA (Immundiagnostik AG, Bensheim, Germany) is used to detect DAO, Zonulin and Calprotectin in serum and stool samples.

The test is performed according to the manufacturer's instructions.
Lactulose/Mannitol test: After overnight fasting, the patient drinks a solution of 100 ml water containing 10g lactulose, 1g D-mannitol and 20g sucrose. Urine is collected over 5 hours while fasting is continued for 3 hours after study start. The urine volume collected during the 5 hours is wrote down and 1ml aliquots are frozen immediately at -80°C with 10% of a 1% thimerosal solution for subsequent analysis by NMR spectroscopy. CpG motifs is used. An increasing concentration of recombinant alkaline phosphatase is added (0, 250 and 500 U/L) to each sample and linear regression is used to account for endogenous alkaline phosphatase in serum.

Endotoxin binding proteins
A ready-to-use solid-phase sandwich ELISA (Hycult biotechnology, Uden, Netherlands) is used to detect LBP and sCD14 levels in EDTA plasma samples.

Neutrophil function
The Phagoburst® kit (Glycotope, Heidelberg, Germany) will be used to determine activation and burst profiles of neutrophils and monocytes by flow cytometric analysis. Cells will be discriminated forward-side-scatter characteristics. Phagocytosis will be assessed by Phagotest® (Glycotope, Heidelberg, Germany), a flow cytometric analysis using FITC-labelled E. coli bacteria.

Statistical analysis
Data will be analyzed with SPSS 21. Data will be described using median and quartiles or percentage of the total group as appropriate. All data will be checked for normal The study participants are healthy volunteers. They will get a compensation of 100 € for completing all study visits to cover their expenses due to the participation in this study. Binge drinking can potentially be harmful, therefore the dose of alcohol chosen is at the lowest limit to fulfil the definition of binge drinking. Furthermore, several safety measures will be applied: Patients will be monitored until they recover; they are not allowed to drive a car on the day of the study and will therefore receive taxi vouchers or need to use public transport and/or need to be accompanied by an adult friend or family member on their way home.
Participants with not benefit directly for the participation in this study. Participants will benefit indirectly from a detailed assessment of their health status. As a further benefit the Principal Investigator of the study will inform each participant individually on the medical risks of binge drinking at visit 2. Taken together in our view the risks and benefits of the study are balanced.
The protocol, informed consent form and the participant information sheet have been submitted to the Ethics Committee of the Medical University of Graz, Austria.
The Investigator will obtain approval from the Ethics Committee of the Medical University of Graz, Austria for all amendments to the original approved documents.

Quality control
The study will be performed in accordance to the ICH-GCP guidelines.