Toll-like receptor 3 dynamics in female C57BL/6J mice: Regulation of alcohol intake

Abstract

Although there are sex differences in the effects of alcohol on immune responses, it is unclear if sex differences in immune response can influence drinking behavior. Activation of toll-like receptor 3 (TLR3) by polyinosinic:polycytidylic acid (poly(I:C)) produced a rapid proinflammatory response in males that increased alcohol intake over time (Warden et al., 2019). Poly(I:C) produced a delayed and prolonged innate immune response in females. We hypothesized that the timecourse of innate immune activation could regulate drinking behavior in females. Therefore, we chose to test the effect of two time points in the innate immune activation timecourse on every-other-day two-bottle-choice drinking: (1) peak activation; (2) descending limb of activation. Poly(I:C) reduced ethanol consumption when alcohol access occurred during peak activation. Poly(I:C) did not change ethanol consumption when alcohol access occurred on the descending limb of activation. Decreased levels of MyD88-dependent pathway correlated with decreased alcohol intake and increased levels of TRIF-dependent pathway correlated with increased alcohol intake in females. To validate the effects of poly(I:C) were mediated through MyD88, we tested female mice lacking Myd88. Poly(I:C) did not change alcohol intake in Myd88 knockouts, indicating that poly(I:C)-induced changes in alcohol intake are dependent on MyD88 in females. We next determined if the innate immune timecourse also regulated drinking behavior in males. Poly(I:C) reduced ethanol consumption in males when alcohol was presented at peak activation. Therefore, the timecourse of innate immune activation regulates drinking behavior and sex-specific dynamics of innate immune response must be considered when designing therapeutics to treat excessive drinking.

Keywords: Alcohol; Alcohol use disorder; Cytokines; Drinking; Females; Neuroimmune; Poly(I:C); Sex; Toll-like receptors.

Figure 1:. Poly(I:C) produces delayed and prolonged innate immune activation in prefrontal cortex of female C57BL/6J mice.

Heat map showing transcript abundance at five time points after poly(I:C) (5mg/kg) injection. The transcript levels are presented using fold-change values (Log2 format) normalized to endogenous control. The red and green colors indicate high and low expression, respectively. The scale representing the relative signal intensity values is shown on the right. Hierarchical clustering was used in the data analysis. (n=6 per group).

Figure 2:. Poly(I:C) decreases alcohol intake when alcohol access occurs at peak innate immune activation in females.

C57BL/6J female mice were injected with saline or poly(I:C) (5mg/kg) every fourth day for a total of ten injections during two-bottle choice, every-other-day drinking (EOD,15%v/v). (A) 4-day injection schedule; (B) Ethanol (EtOH) intake (g/kg/24 h); (C) preference for EtOH; (D) total fluid intake. Arrows indicate days when animals received injection; (n=10 per group).

Figure 3:. Poly(I:C) prevents reductions in alcohol intake when alcohol access occurs after peak innate immune activation in females.

C57BL/6J female mice were injected with saline or poly(I:C) (5mg/kg) every fifth day for a total of ten injections during two-bottle choice, every-other-day drinking (EOD,15%v/v). (A) 5-day injection schedule; (B) Ethanol (EtOH) intake (g/kg/24 h); (C) preference for EtOH; (D) total fluid intake. Arrows indicate days when animals received injections; (n=10 per group).

Figure 4:. Innate immune transcript abundance correlates with directional changes in alcohol intake in females.

(A) Heat map showing transcript abundance after both the 4-day poly(I:C) injection schedule and 5-day poly(I:C) injection schedule drinking experiments. The transcript levels are presented using fold-change values (Log2 format) normalized to endogenous control and saline-treated controls for each injection schedule. (B) Correlation heatmap of individual transcripts with average ethanol intake. The red and green colors indicate high and low transcript abundance or correlation strength, respectively. For the 4-day injection schedule, there were significant positive correlations between Myd88 (C) and amount of ethanol consumed, between Ikkβ (D) and amount of ethanol consumed and Ifnar1 (E) and amount of ethanol consumed in poly(I:C)-treated mice. For the 5-day injection schedule, there were significant positive correlations between Ticam1 (F) and amount of ethanol consumed and between Ikkε (G) and amount of ethanol consumed in poly(I:C)-treated mice. ****p<0.0001, ***p<0.0002, **p<0.0021, *p<0.05 for Tukey post-hoc tests and correlations.

Figure 5:. Poly(I:C)-induced decreases alcohol intake are dependent on MyD88.

Mutant female mice (Myd88 −/−) were injected with saline or poly(I:C) (5mg/kg) every four days for a total of seven injections during a two-bottle choice every-other-day drinking procedure (EOD, 15%v/v). (A) Ethanol (EtOH) intake (g/kg/24 h), (B) preference for EtOH, and (C) total fluid intake (n=10 per group).

Figure 6:. Poly(I:C) does not change saccharin taste perception.

C57BL6/J female mice were injected with saline of poly(I:C) (5mg/kg) every four days for a total of four injections during a two-bottle-choice every-other-day saccharin procedure (EOD, 0.008%). (A) Saccharin consumption (g/kg/24 h), (B) preference for saccharin, and (C) total fluid intake (g/kg/24 h) (n=10 per group).

Figure 7:. Poly(I:C) prevents escalation of alcohol intake when alcohol access occurs at peak innate immune activation in males.

C57BL/6J male mice were injected with saline or poly(I:C) (5mg/kg) every fourth day for a total of eight injections during two-bottle choice, every-other-day drinking (EOD,15%v/v). (A) 3hr delay injection schedule; (B) Ethanol (EtOH) intake (g/kg/24 h); (C) preference for EtOH; (D) total fluid intake. Arrows indicate days when animals received injection; (n=10 per group).

Figure 8:. Males and females have different innate immune activation timecourses.

Expression of Il6 mRNA after poly(I:C) (5mg/kg) injection. Note after poly(I:C), male peak innate immune activation occurs between 3-8 hours post-injection and female peak innate immune activation occurs between 24-48 hours post-injection. mRNA was normalized to saline treated animals as well as the endogenous control, Gusb.