Neuroimmune regulation of alcohol consumption: behavioral validation of genes obtained from genomic studies

Abstract

Analysis of mouse brain gene expression, using strains that differ in alcohol consumption, provided a number of novel candidate genes that potentially regulate alcohol consumption. We selected six genes [beta-2-microglobulin (B2m), cathepsin S (Ctss), cathepsin F (Ctsf), interleukin 1 receptor antagonist (Il1rn), CD14 molecule (Cd14) and interleukin 6 (Il6)] for behavioral validation using null mutant mice. These genes are known to be important for immune responses but were not specifically linked to alcohol consumption by previous research. Null mutant mice were tested for ethanol intake in three tests: 24-hour two-bottle choice, limited access two-bottle choice and limited access to one bottle of ethanol. Ethanol consumption and preference were reduced in all the null mutant mice in the 24-hour two-bottle choice test, the test that was the basis for selection of these genes. No major differences were observed in consumption of saccharin or quinine in the null mutant mice. Deletion of B2m, Ctss, Il1rn, Cd14 and Il6 also reduced ethanol consumption in the limited access two bottle choice test for ethanol intake; with the Il1rn and Ctss null mutants showing reduced intake in all three tests (with some variation between males and females). These results provide the most compelling evidence to date that global gene expression analysis can identify novel genetic determinants of complex behavioral traits. Specifically, they suggest a novel role for neuroimmune signaling in regulation of alcohol consumption.

Figure 1. Lack of B2m, Ctss, Ctsf, Il1rn, Il6 or Cd14 reduces ethanol preference in two-bottle choice test in male mice

A. B2m knockout mice, (p<0.01, effect of genotype). B. Ctss knockout mice, (p<0.001, effect of genotype). C. Ctsf knockout mice. (p<0.05, effect of genotype). D. Il1rn knockout mice, (p<0.001, effect of genotype). E. Cd14 knockout mice, (p<0.001, effect of genotype). F. Il6 knockout mice, (p<0.001, effect of genotype). n = 9 – 10 for all groups.

Figure 2. Lack of B2m, Ctss, Il1rn, Il6 or Cd14 reduces ethanol preference in two-bottle choice test in female mice

A. B2m knockout mice, (p<0.01, effect of genotype). B. Ctss knockout mice, (p<0.001, effect of genotype). C. Ctsf knockout mice, (p>0.05, effect of genotype). D. Il1rn knockout mice, (p<0.001, effect of genotype). E. Cd14 knockout mice, (p<0.001, effect of genotype). F. Il6 knockout mice, (p<0.001, effect of genotype). n = 9 – 10 for all groups.

Figure 3. Effect of deletion of B2m, Ctss, Il1rn, Cd14 or Il6 on preference for ethanol in two-bottle DID procedure (2B-DID)

Males: A. B2m and Ctss knockout mice. B. Il1rn knockout mice. (p<0.001, effect of genotype). C. Cd14 and Il6 knockout mice. Females: D. B2m and Ctss knockout mice. (p<0.05, effect of genotype for the Ctss knockout mice) E. Il1rn knockout mice. (p<0.001, effect of genotype) F. Cd14 and Il6 knockout mice. (p<0.01, effect of genotype for the Cd14 knockout mice) n = 10 – 14 for all groups. Ethanol was used in concentration 20%.

Figure 4. Effect of deletion of B2m, Ctss, Il1rn, Cd14 or Il6 on preference for ethanol in one bottle DID procedure (1B-DID) in male mice

A. B2m knockout mice. B. Ctss knockout mice, (p<0.001, effect of genotype for the period with 2 hrs access). C. Il1rn knockout mice, (p<0.001, effect of genotype for the period with 2hrs access). D. Cd14 knockout mice. E. Il6 knockout mice. ** - p<0.01, *** - p<0.001 – statistically significant differences between mutant and correspondent wild type mice for the period with 4 hrs access (Student’s t-test). n = 7 – 9 for all groups. Ethanol was used in concentration 20%.

Figure 5. Effect of deletion of B2m, Ctss, Il1rn, Cd14 or Il6 on preference for ethanol in one bottle DID procedure (1B-DID) in female mice

A. B2m knockout mice. B. Ctss knockout mice, (p<0.001, effect of genotype for the period with 2hrs access). C. Il1rn knockout mice, (p<0.001, effect of genotype for the period with 2hrs access). D. Cd14 knockout mice. E. Il6 knockout mice. *** - p<0.001 – statistically significant differences between mutant and correspondent wild type mice for the period with 4 hrs access (Student’s t-test). n = 6 – 8 for all groups. Ethanol was used in concentration 20%.

Figure 6

Schematic diagram linking genes that were targeted for functional validation using null mutant mice. The diagram is based on existing pathways from KEGG (http://www.genome.jp/kegg/) and BioCarta (http://www.biocarta.com/) as well as published literature (see Discussion for references). Cd14 is a key component of the Toll-like receptor pathway resulting in production of proinflammatory cytokines, such as IL-1 and IL-6. IL1RN is an antagonist for IL-1 receptor. Cathepsins S and F are cysteine proteases that degrade the invariant polypeptide of major histocompatibility complex, class II (Cd74) and promote the presentation of antigens by antigen-presenting cells. This function is served by microglia and astrocytes in brain. B2m also plays a role in antigen presentation.