Alcohol Use Disorder and Inflammatory Cytokines in a Population Sample of Young Adults

Background: Alcohol abuse is followed by neuroadaptive brain changes, in addition to inducing changes in the immune system. The objective of this study was to investigate the peripheral levels of proinflammatory (IL-6, and TNF-α) and anti-inflammatory (IL-10) cytokines, as well the inflammatory balance in relation to alcohol use in subjects from a young population-based sample. Methods: This is a cross-sectional study nested in a population-based study of people aged 18–35 years, involving 629 participants. The CAGE questionnaire was used to evaluate Alcohol Use Disorder, and CAGE scores ≥ 2 were considered a positive screen for alcohol use disorder. Serum levels of IL-6, IL-10 and TNF-α were measured by ELISA using a commercial kit. Results: There was as a statistically significant increase in IL-6 (p ≤ 0.001) and statistically significant decrease in IL-10 (p=0.017) serum levels in the Alcohol Use Disorder group when compared with subjects that did not abuse alcohol and that a CAGE score <2. TNF-α levels were not significantly different. There was a statistically significant difference in IL-6/IL-10 (p ≤ 0.001). The median ratios were 0.28 (0.20 to 0.36) in the CAGE group with scores <2 and 0.70 (0.52 to 0.93) in the group with alcohol use disorder for the IL-6/IL-10. Conclusion: Our results suggest that cytokines, especially IL-6 and IL-10, are involved in the pathophysiology of abuse and dependence of alcohol, and could be candidate markers of alcohol use disorder.


Introduction
Alcohol consumption is widespread throughout the world and it results in numerous negative consequences for health and quality of life, especially in the young population [1][2][3][4]. Alcohol abuse is responsible for 3.2% of all deaths, placing it next to tobacco as a major preventable cause of death and disability [5]. Alcohol intake is followed by neuroadaptive changes; in addition, it induces changes in the immune system, increasing the risk of infections [6]. Nowadays, the diagnosis of alcoholism is based on clinical signs and peripheral injury markers, which, unfortunately, are not specific or sensitive enough to determine the effects of alcohol abuse on central nervous system (CNS) [7,8].
Alcohol abuse has been described to increase immune activity in the CNS, contributing to the process of neurodegeneration and impaired neurological function [9]. The immune system plays an important role in the CNS related to survival and neuronal death [10,11]. In recent years, many studies have suggested the involvement of inflammatory cytokines in the pathophysiology of several neurological and psychiatric disorders such as alcoholism [12]. Abuse of alcohol induces microglia to release proinflammatory cytokines, such as IL-6 and TNFα, increasing inflammation and neuronal damage [13,14]. To reduce the release of proinflammatory cytokines, the immune system releases immunosuppressive cytokines, such as IL-10. However, imbalance in cytokine levels can lead to insufficient mediation or inhibition of normal immune reaction to disease manifestation [15]. This has been recently documented in animal models of chronic alcohol consumption showing an increase in proinflammatory cytokines and a decrease in anti-inflammatory cytokines (IL-10) [9,16]. Therefore, these studies indicate that changes in inflammatory balance may be involved in alcoholism. Alcohol use disorders are usually underdiagnosed and present major difficulties regarding monitoring and treatment. The combination of multiple biomarkers may significantly increase the sensitivity and specificity of the biochemical tests, putatively helping in early detection and, consequently, therapeutic success. Given the role of cytokines in chronic alcohol exposure, the objective of this study was to investigate the peripheral levels of proinflammatory (IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines, as well the inflammatory balance in relation to alcohol abuse in subjects from a young population-based sample compared to subjects that did not abuse alcohol

Materials and Method
This is a cross-sectional study nested in a population-based study of people aged 18-35 years, involving 629 participants living in the city of Pelotas, Brazil. The study was approved by the Catholic University of Pelotas's Ethics Committee (2010/15). After the subjects were identified and invited, volunteers signed an informed consent and answered to a questionnaire, which collected sociodemographic data, drug misuse and The National Economic Indicator-IEN [17]. To evaluate alcohol use disorder, the participants also responded to the CAGE questionnaire, which asks about four consequences of drinking. CAGE is a validated screening test for alcohol abuse and dependence [18]. Briefly, patients score 1 point for each "yes" on the CAGE and "0" points if all questions are answered "no". A score of 2 or more (CAGE ≥ 2) is usually considered a positive screen for alcohol use disorder. A score below 2 points (CAGE<2) constituted the subjects did not abuse alcohol [19].
For the biochemical analyses, 10 millilitres of blood were withdrawn from each subject by venepuncture into an anticoagulant-free vacuum tube after the interview. The blood was immediately centrifuged at 4,000 rpm for 10 min, and the serum was kept frozen at -80°C until analyses. Serum levels of IL-6, IL-10, TNF-α were measured using a commercial immunoassay kit (Duo Set ELISA Development, R&D Systems, Inc., USA). IL-6, IL-10, TNF-α serum levels were expressed in pg/mL. The ratio of pro-and anti-inflammatory cytokines (IL-6/IL-10 ratio) provided an index for the balance between pro and antiinflammatory status, and the ratio was considered as an index of proinflammatory activity. Statistical analyses were performed with the Statistical Program for Social Sciences (SPSS) 21.0 and Graph Pad Prism 6 software's. IL-6, IL-10, TNF-α serum levels had non-Gaussian distributions. For nonparametric data, we utilized the Mann-Whitney and Kruskal-Wallis tests and Spearman correlation. Serum levels of cytokines were presented as median and interquartile range. A linear regression analysis was applied to control for possible confounding factors with a p value ≤ 0.2 in the bivariate analyses. Results with p values ≤ 0.05 were considered statistically significant.

Results
Among the 629 analyzed subjects, 70 (11.1%) were identified with alcohol use disorder. Table 1 shows the sociodemographic characteristics and serum levels of cytokines (IL-6, IL-10 and TNF-α).  There was a statistically significant difference between groups in IL-6 (p ≤ 0.001) and IL-10 (p=0.017) serum levels. IL-6 levels were higher in the alcohol use disorder group, with a median of 22.07 (16.62 to 43.66) pg/mL, in relation to the group with CAGE scores <2, which had a median of 15.40 (10.78 to 21.27) pg/mL. In addition, levels of IL-10 were lower in the alcohol use disorder group, with a median of 33.81 (21.28 to 80.40) pg/mL, compared to subjects that did not abuse alcohol, which had a median of 56.42 (47.51 to 69.24) pg/mL. For TNF-α levels, there was no significant difference (p=0.788) ( Table 1).
In the alcohol abuse disorder group, 34.3% (n=24) of individuals presented depression and 30% (n=21) were anxious. We did not find a significant association between alcohol abuse disorder and depression (p=0.112), however, there was a trend for anxiety (p=0.082). Furthermore, there were no significant differences between socioeconomic terciles (p=0.748) and alcohol abuse disorder.

Discussion
In this study, we evaluated cytokine levels related to alcohol use disorder in young subjects. Subjects with alcohol use disorder showed higher IL-6 serum levels and lower IL-10 serum levels, with no change in TNF-α serum levels when compared with subjects that did not abuse alcohol. Moreover, the IL-6/IL-10 ratio was significant.
Cytokines have been increasingly recognized as a signal molecule regulating the homeostatic responses of several tissues, and the deleterious effects of alcohol abuse on the immune system are widely recognized [16]. Furthermore, several studies have suggested that imbalance in the immune system can reflect negatively in the brain function and behavior, suggesting that these effects may be implicated in neuropsychiatric disorders in alcoholic individuals [20]. In this sense, several studies have shown an increase in proinflammatory cytokines in alcohol-dependent patients [21][22][23], suggesting a possible involvement of these molecules in alcohol abuse disorder [21]. In this study, we suggest that the increase of proinflammatory cytokines may be indicative of alcohol abuse disorder, as we found similar results to those in the literature that supports our hypothesis. Likewise, Gonzalez-Quintela [24] observed increased serum concentrations of cytokines (IL-6 and IL-10) in alcoholic patients. After a few days of withdrawal, there was a significant decrease. However, levels remained elevated when compared with healthy subjects [24].
Imbalance of the inflammatory state induced by alcohol abuse leads to several physiological and behavioral changes [9]. Several studies have shown that the inflammatory system is closely related to the emergence of various mood disorders [25][26][27][28]. Immune system activation affects several neural pathways, particularly the neurotransmission and expression of trophic factors [29]. Excessive alcohol consumption leads to increased levels of inflammatory cytokines in the central and peripheral nervous systems [9]. Because peripheral system cytokines may cross the blood-brain barrier, the increase of cytokines in the CNS leads to detrimental effects on the brain, contributing to alterations in neurological function and neurodegeneration [30]. Given the fundamental role of neurotrophins in neurogenesis and synaptic plasticity, the inflammatory process may affect brain function, potentially altering Brain-derived neurotrophic factor (BDNF) modulation [31,32]. Several studies have shown that inflammatory response in the brain can change BDNF expression, which may compromise the process of neuroplasticity [33,34]. In a previous study, we found that individuals who abused alcohol had increased levels of neurotrophic factors (BDNF, NGF and GDNF) [35].
Alcohol abuse is closely related to increased morbidity and mortality, and has been specially associated with psychiatric disorders [36]. Moreover, alcohol is often used as "self-medication" in patients with psychiatric disorders, as it is reported to temporarily alleviate feelings of anxiety and depression [37]. In our study, no differences were observed in the prevalence of mood disorders among individuals who abused and did not abuse alcohol. changes in the brain [38]. Alcohol-induced liver damage generates inflammatory cytokines, which may influence brain cells [39], and thus alter brain functions [38]. The neuroinflammation process is accompanied by an increase of reactive oxygen species and inflammatory proteases, causing cellular damage and microglia activation [38,40]. It is theorized that high concentrations of cytokines in the CNS that are associated with heavy alcohol consumption can remain active for an extended period of time, thereby altering neurotransmission, neurogenesis, neurite outgrowth, myelination, and may promote neurodegeneration [38,41,42]. Thus, alcohol-induced immune system changes can be relevant in the development of neuropathological and behavioral changes [12,38]. In addition, alcoholic patients without cirrhosis have presented increase in IL-6 levels [22]. In fact, reliable markers may be useful to confirm the diagnosis and the current stage of the disease, especially with the uncertain information provided by patients [43,44]. Therefore, abusive alcohol consumption increases proinflammatory release, promoting, supposedly, the appearance of mental disorders. In this study, we cannot evaluate the frequency and amount of intake in individuals who abused alcohol and also the period of abstinence. Also, we were unable to assess the stage of the disorder. However, our study differs from other literature because it involves young individuals from a population-based study; they were not making use of medication and they were not admitted into clinics for treatment of alcohol abuse or other disorders. In order to find new biomarkers to aid diagnosis in clinical assessment, treatment and monitoring alcohol use disorders, our results suggest that inflammatory parameters, such as IL-10 and IL-6 may be involved in the pathophysiology of this disorder. However, more studies are needed to assess inflammatory status in the different stages of this disorder.
The search for biological and clinical markers for psychiatric disorders is highly relevant for diagnosis, treatment, and monitoring of these disorders, thus enabling higher precision and agility in the diagnosis. Current markers (liver enzymes and hematological changes) of alcoholism are only useful when the patient has already been in heavy alcohol usage for quite a while, so it is necessary to find new biological parameters for the early diagnosis of alcohol use disorder [45]. Our results suggest that cytokines, especially IL-6 and IL-10, are involved in the pathophysiology of alcohol abuse/dependence. Moreover, they may be candidate markers of neuronal damage. However, these actions are complex and the mechanisms responsible for ethanol's effect on immune responses remain elusive. More studies are needed to better assess the profile of cytokines in alcohol use disorder.