Chronic excessive alcohol consumption leads to neuroinflammation and may result in cognitive dysfunction and behavioural changes, as alcohol rapidly diffuses through the blood-brain barrier, alters neurotransmission, contributes to neurodegeneration, and impairs regeneration by activating microglia and astrocytes, but our understanding of the mechanisms by which alcohol triggers inflammation in the brain is still limited (1). What we do know is that peripheral endotoxemia induced by alcohol may lead to increased secretion of pro-inflammatory cytokines such as TNF-α, interleukin (IL)-1β, IL-6, and interferon gamma (2). We also know that astrocyte activation may be mediated by the Toll-like receptor 4 pathway (TLR4), which activates downstream signalling molecules and cytokine secretion (1, 3). One study on mice has shown that ethanol directly triggers TLR4-mediated activation of the nucleotide-binding oligomerisation domain (NOD), leucine-rich repeats (LRR), and pyrin domain-containing protein 3 (NLRP3) inflammasome in glia cells (4). Another study on human and mouse cells suggests that the hyper-activation of the NLRP3 inflammasome may also be related to prolonged exposure to the products of ethanol metabolism (5).
The NLRP3 inflammasome is a cytoplasmic complex of intracellular sensors such as NOD-like receptors coupled with procaspase-1 and the apoptosis-associated speck-like protein containing a caspase-associated recruitment domain (ASC) (6). Molecular damage triggers the assembly of the NLRP3 inflammasome leading to and IL-1β secretion and caspase-1 activation (7), which has been associated with early development of atherosclerosis in cerebral vessels and other heritable and acquired inflammatory diseases (8).
As
However, up to date no human study has investigated the association between NLRP3 polymorphisms and alcohol dependence, and the aim of our study was to fill that gap by investigating the association between the above two polymorphisms, namely
The study included only male participants (to exclude the influence of sex differences, see ref. 22) aged from 18 to 65 years from the Slovenian (Caucasian) population. The first group (group 1; N=88) included hospitalised alcohol-dependent patients who met the criteria of alcohol dependence of the 4th edition of Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) (23) and were no longer having major abstinence symptoms (after having spent at least two weeks in respective departments/units for addiction treatment). The second group (group 2; N=99) included abstinent alcohol-dependent individuals recruited from support group meetings who had maintained full abstinence for more than two years. Participants of either group were excluded if they had a history of abuse or dependence of psychoactive substances other than nicotine and other major mental or neurological disorders or significant medical illnesses in their medical records. The third group (group 3; N=94) included healthy controls recruited from blood donors, who answered a short structured clinical questionnaire through interview to exclude DSM-IV Axis I disorders or clinical problems with alcohol consumption.
All participants also gave information about their residence (rural or urban), partnership status (single or in partnership: either married or living in the extramarital union), and years of education and signed informed consent.
The following questionnaires were employed: Zung Depression (24) and Anxiety (25) scale and Brief Social Phobia Scale (BSPS) (26) to rate depression and anxiety symptoms, Alcohol Use Disorders Identification Test (AUDIT) (27) to define drinking habits and severity of alcohol problems and dependence, Yale-Brown Obsessive Compulsive Scale (YBOCS) (28) and Obsessive Compulsive Drinking Scale (OCDS) (29) to rate obsessive-compulsive traits, and Buss-Durkee Hostility Inventory (BDHI) (30) to rate symptoms of aggression and hostility. All the questionnaires were administered at the entry into the study by the same rater, who was blinded to the genotyping results.
The study was approved by the Slovenian National Medical Ethics Committee (approval No. 117/06/10 and 148/02/1011) and followed the latest version of the Declaration of Helsinki (31). Each participant received a code to protect all the personal and medical information. All biological and DNA samples were processed and analysed under these codes.
DNA was isolated from either 5 mL of whole blood collected by venepuncture from groups 1 and 3 (hospitalised patients and healthy controls, respectively) or from a buccal swabs collected from group 2 (abstinents). Whole blood was collected into ethylenediaminetetraacetic acid (EDTA) tubes as part of regular blood testing (group 1) or donation (group 3) and stored at +4 °C until extraction. For DNA extraction from blood samples we used the QIAamp Blood Mini Kit and for the extraction from buccal swabs the QIAamp Mini Kit (Qiagen GmbH, Hilden, Germany) following manufacturer’s instructions (32).
Pearson’s chi-squared test was used to compare SNP frequencies between the three groups and their effects on categorical variables, i.e. total scores on applied questionnaires. One-way analysis of variance (ANOVA) was used to assess SNP effects and continuous variables in each of the three groups separately. The associations between each SNP and continuous variables (genotype-phenotype associations) were compared between the groups with the factorial ANOVA. The level of statistical significance was set at 0.05. The study had sufficient power (0.80) to detect small-to-medium effect sizes (f2=0.021 and d=0.355). All statistics were run on the Statistica package, version 7.0 for Windows® (StatSoft Italia, Vigonza, Padua, Italy).
Table 1 shows participant demographic characteristics of interest. Hospitalised and abstinent alcohol-dependent patients were significantly older (P<0.001; df=2; F=46.080) and had fewer years of education than controls (P<0.001; df=2; F=46.080). Significantly more abstinent alcohol dependents and controls were in partnership than hospitalised alcohol dependents (P=0.004; df=2; F=5.601).
Socio-demographic characteristics of study participants
Characteristics | Hospitalised alcohol- dependent patients (n=88) | Abstinent alcohol-dependent participants (n=99) | Controls (n=94) | p-value |
---|---|---|---|---|
Age (years) | 45.8±10.0 | 49.1±8.1 | 34.4±11.7 | 0.003 |
Education (years) | 11.3±2.2 | 11.7±2.4 | 12.7±1.9 | 0.001 |
Single | 38 (43 %) | 22 (22 %) | 24 (25 %) | 0.004 |
In partnership | 50 (56 %) | 77 (78 %) | 70 (75 %) | |
Rural residents | 45 (51 %) | 48 (48 %) | 36 (38 %) | 0.293 |
Urban residents | 43 (49 %) | 51 (52 %) | 58 (62 %) |
Means ± standard deviations are given for continuous or the number of participants for categorical variables. Partnership means marital or extramarital union. p-value is shown for the comparison between all three groups
Genotype distribution of
Genotypes | Hospitalised alcohol-dependent patients (n=88) | Abstinent alcohol- dependent participants (n=99) | Controls (n=94) | p-value1 | Merged alcohol- dependents (n=185) | p-value2 |
---|---|---|---|---|---|---|
0.838 | 0.500 | |||||
CC | 78 (88 %) | 87 (88 %) | 87 (93 %) | 165 (88 %) | ||
AC | 9 (11 %) | 11 (11 %) | 6 (6 %) | 20 (11 %) | ||
AA | 1 (1 %) | 1 (1 %) | 1 (1 %) | 2 (1 %) | ||
0.055 | ||||||
AA | 34 (39 %) | 52 (53 %) | 45 (48 %) | 86 (46 %) | ||
AT | 44 (51 %) | 42 (42 %) | 34 (36 %) | 86 (46 %) | ||
TT | 9 (10 %) | 5 (5 %) | 15 (16 %) | 14 (8 %) |
p-1 comparison between all three groups. p-2 comparison between the merged groups of alcohol-dependents and controls. Bolded p-values are statistically significant (p<0.05)
No significant association was found between the
Associations between
Mental disorders | Hospitalised alcohol-dependent patients | Abstinent alcohol-dependent participants | Controls | ||||
---|---|---|---|---|---|---|---|
Mean score±SD | p-value | Mean score±SD | p-value | Mean score±SD | p-value | ||
CC | 4.2±4.6 | 0.742 | 1.8±2.2 | 0.661 | 1.7±1.5 | 0.498 | |
AC | 3.6±4.4 | 1.3±2.5 | 1.0±0.0 | ||||
AA | 1.0 | 1.0 | 1.0 | ||||
CC | 2.9±3.1 | 0.787 | 1.4±1.8 | 0.810 | 1.3±0.9 | 0.644 | |
AC | 3.2±3.3 | 1.1±0.3 | 1.0±0.0 | ||||
AA | 1.0 | 1.0 | 1.0 | ||||
CC | 12.9±11.0 | 0.555 | 12.1±10.1 | 0.353 | 10.5±7.2 | 0.615 | |
AC | 8.8±10.9 | 16.5±11.1 | 8.7±1.3 | ||||
AA | 10.0 | 18.0 | 5.0 | ||||
CC | 18.9±11.1 | 0.681 | 3.2±2.3 | 0.757 | 3.5±1.7 | 0.926 | |
AC | 15.8±13.2 | 3.7±3.3 | 3.3±0.8 | ||||
AA | 14.0 | 3.0 | 4.0 | ||||
Depression (Zung) | CC | 35.9±11.0 | 0.881 | 31.1±7.4 | 0.653 | 23.0±3.8 | 0.311 |
AC | 34.9±11.5 | 29.3±6.1 | 21.8±2.4 | ||||
AA | 31.0 | 27.0 | 28.0 | ||||
CC | 34.9±8.4 | 0.535 | 30.1±6.9 | 0.886 | 22.6±3.1 | 0.742 | |
AC | 31.9±7.1 | 31.1±5.6 | 22.7±3.4 | ||||
AA | 31.0 | 29.0 | 25.0 | ||||
CC | 24.3±10.8 | 0.853 | 20.5±10.3 | 0.735 | 14.8±8.8 | 0.220 | |
AC | 26.0±7.2 | 19.6±7.4 | 10.2±4.1 | ||||
AA | 21.0 | 13.0 | 25.0 | ||||
CC | 23.0±6.8 | 0.069 | |||||
AC | 28.4±4.0 | ||||||
AA | 23.0 |
AUDIT – Alcohol Use Disorders Identification Test; BDHI – Buss-Durkee Hostility Inventory; BSPS – Brief Social Phobia Scale; OCDS – Obsessive Compulsive Drinking Scale; YBOCS – Yale-Brown Obsessive Compulsive Scale
Associations between
Mental disorder | CARD8 genotype | Hospitalised alcohol-dependent patients | Abstinent alcohol-dependent subjects | Controls | |||
---|---|---|---|---|---|---|---|
Mean score±SD | p-value | Mean score±SD | p-value | Mean score±SD | p-value | ||
AA | 3.6±4.0 | 0.244 | 1.4±1.9 | 0.203 | 1.5±1.3 | 0.591 | |
AT | 2.7±5.1 | 2.0±2.0 | 1.8±1.7 | ||||
TT | 2.2±2.2 | 2.8±4.0 | 1.8±1.3 | ||||
AA | 2.9±3.5 | 0.481 | 1.3±1.9 | 0.960 | 1.2±0.6 | 0.406 | |
AT | 3.0±2.9 | 1.5±1.3 | 1.4±1.2 | ||||
TT | 1.7±1.7 | 1.4±0.9 | 1.3±0.6 | ||||
AA | 10.2±8.8 | 0.291 | 11.7±8.2 | 0.567 | 10.9±7.7 | 0.695 | |
AT | 12.7±11.5 | 13.9±12.3 | 10.2±7.0 | ||||
TT | 16.0±10.3 | 13.2±10.8 | 9.1±4.4 | ||||
AA | 16.2±11.6 | 0.161 | 3.5±2.7 | 0.401 | 3.5±1.4 | ||
AT | 18.9±10.9 | 2.9±2.0 | 3.9±2.0 | ||||
TT | 24.1±10.8 | 2.5±0.9 | 2.6±0.8 | ||||
AA | 34.1±8.3 | 0.560 | 30.0±6.7 | 0.418 | 23.5±4.3 | 0.201 | |
AT | 36.6±11.7 | 31.5±8.0 | 22.1±3.3 | ||||
TT | 37.1±16.3 | 33.6±6.5 | 23.4±3.7 | ||||
AA | 32.9±6.2 | 0.320 | 29.1±5.6 | 23.0±3.5 | 0.477 | ||
AT | 35.5±9.1 | 30.8±7.4 | 22.1±2.7 | ||||
TT | 36.1±10.6 | 36.4±9.5 | 22.5±2.5 | ||||
AA | 26.0±8.7 | 0.534 | 19.8±8.3 | 0.758 | 15.0±9.0 | 0.970 | |
AT | 23.3±11.6 | 20.7±11.9 | 14.2±9.3 | ||||
TT | 23.4±11.6 | 23.0±10.3 | 14.3±6.5 | ||||
AA | 23.4±6.5 | 0.818 | |||||
AT | 23.9±7.2 | ||||||
TT | 22.3±5.4 |
Bolded p-values are statistically significant (p<0.05). AUDIT – Alcohol Use Disorders Identification Test; BDHI – Buss-Durkee Hostility Inventory; BSPS – Brief Social Phobia Scale; OCDS – Obsessive Compulsive Drinking Scale; YBOCS – Yale-Brown Obsessive Compulsive Scale
We found that
Another significant association we did observe is the one between
The limitation of our study is a relatively small but ethnically homogeneous sample. The advantage is that all the questionnaires were applied by the same rater.
In conclusion, our findings point to a link between the genes of the innate immune system and alcohol dependence. However, further studies with larger cohorts are required to confirm these preliminary findings.