Evaluation of TLR4 Inhibitor, T5342126, in Modulation of Ethanol-Drinking Behavior in Alcohol-Dependent Mice

Abstract

Aims: Several lines of evidence support a critical role of TLR4 in the neuroimmune responses associated with alcohol disorders and propose inhibitors of TLR4 signaling as potential treatments for alcoholism. In this work, we investigated the effect of T5342126 compound, a selective TLR4 inhibitor, on excessive drinking and microglial activation associated with ethanol dependence.

Methods: We used 2BC-CIE (two-bottle choice-chronic ethanol intermittent vapor exposure) paradigm to induce ethanol dependence in mice. After induction of the ethanol dependence, we injected T5342126 (i.p., 57 mg/kg) for 14 days while monitoring ethanol intake by 2BC (limited access to ethanol) method.

Results: T5342126 decreased ethanol drinking in both ethanol-dependent and non-dependent mice but T5342126 showed also dose-dependent non-specific effects represented by decreased animal locomotor activity, saccharine intake, and body core temperature. Six days after the last ethanol-drinking session, we examined the immunohistochemical staining of Iba-1 (ionized calcium-binding adapter molecule 1), a microglial activation marker, in the central nucleus of the amygdala (CeA) and dentate gyrus (DG) of the hippocampus. Notably, T5342126 reduced Iba-1 density in the CeA of both ethanol-dependent and non-dependent mice injected with T5342126. There were no significant differences in the DG Iba-1 density among the treatment groups.

Conclusions: Collectively, our data suggest that T5342126, via blocking TLR4 activation, contributes to the reduction of ethanol drinking and ethanol-induced neuroimmune responses. However, the non-specific effects of T5342126 may play a significant role in the T5342126 effects on ethanol drinking and thus, may limit its therapeutic potential for treatment of alcohol dependence.

Short summary: T5342126, an experimental TLR4 inhibitor, is effective in reducing ethanol drinking and inhibiting the activation and proliferation of microglia in both ethanol-dependent and non-dependent mice. However, T5342126's use as a potential candidate for the treatment of alcohol addiction may be limited due to its non-specific effects.

Fig. 1.

Experimental design and blood alcohol levels (BAL) in ethanol-dependent mice. (A) Overview of the 2BC-CIE treatment and the T5342126 injection paradigm. We injected T5342126 intra-peritoneally 30 min prior to 2BC testing for 15 consecutive days (injection 1 = 82 mg/kg; injections 2–15 = 57 mg/kg). (B) The chemical structure of T5342126. (C) The mean BAL were not significantly different between the mice selected for vehicle and T5342126 injections (two-way RM ANOVA, F_(1,38) = 0.94; P = 0.34). The BAL varied significantly between the vapor bouts (F_(2,38) = 3.88; P = 0.03).

Fig. 2.

T5342126 reduced ethanol and saccharin drinking in both control and ethanol-dependent mice. (A) The average daily ethanol intake of control and ethanol-dependent (CIE) mice injected with vehicle or T5342126 during measured by 2BC during limited 2 h access to ethanol (15%). (B) The baseline ethanol drinking, measured prior to T5342126 injections, was significantly increased in the ethanol-dependent mice compared to control mice (F_(1,40) = 15.55, ^###P = 0.0003). There were no significant differences in ethanol drinking between the mice selected for vehicle and T5342126 injections within the ethanol-vapor-treated groups (F_(1,40) = 0.13, P > 0.05). (C–E) The average weekly ethanol intake of the control and ethanol-dependent (CIE) mice following T5342126 injections. Two-way ANOVA showed significant main effects of ethanol-vapor exposure and T5342126 injections in week 1 (vapor exposure: F_(1,40) = 13.25, ^##P = 0.0008; T5342126 injection: F_(1,40) = 6.57, ^P = 0.014) and week 2 (vapor exposure: F_(1,39) = 12.33, ^##P = 0.0011; T5342126 injection: F_(1,39) = 8.37, ^^P = 0.0062) that were not associated with significant interaction between the factors. In week 3 of the 2BC testing (E), we found significant main effect of the T5342126 injections (F_(1,39) = 8.27, ^^P = 0.0065) but not of the ethanol-vapor exposure (F_(1,39) = 2.76, P > 0.05). (F) Two days after the last T5342126 injection (drug clearance), we found no significant differences in ethanol intake between the vehicle and T5342126-injected control or ethanol-dependent mice. (G) The T5342126 injections significantly decreased saccharin intake in both control and ethanol-dependent mice (F_(1,36) = 6.15, ^P = 0.018). We did not observe significant differences in saccharin intake between control and ethanol-dependent mice (F_(1,36) = 1.09, P = 0.304). The data are presented as mean + SEM.

Fig. 3.

T5342126 reduced water consumption in both control and ethanol-dependent mice. The average daily water consumption measured by 2BC during 2 h testing period. (A) There was no significant difference in baseline water intake between the T5342126- and vehicle-injected ethanol-dependent (CIE) and non-dependent (control) mice (main effect of ethanol-vapor exposure: F_(1,40) = 0.00; main effect of T5342126 injections: F_(1,40) = 0.01). (B) Week 1 of the T5342126 injections: Neither ethanol-vapor exposure nor T5342126 have significant effect on the water consumption measured by 2BC testing in the first week T5342126 injections (main effect of ethanol-vapor exposure: F_(1,40) = 2.14; main effect of T5342126 injections: F_(1,40) = 2.86). (C) Week 2 of the T5342126 injections: T5342126 had significant main effects on the water consumption in both CIE and control mice (main effect of T5342126 injections: F_(1,39) = 4.43, P = 0.042), while ethanol-vapor exposure had no significant impact on the water consumption (main effect of ethanol-vapor exposure: F_(1,40) = 0.21). (D) Week 3 of T5342126 injections: The water consumption was decreased in T5342126-injected mice compared to vehicle-injected mice (F_(1,36) = 4.17, P = 0.049). Ethanol-vapor exposure did not significantly altered the water consumption (F_(1,36) = 3.12). The data are presented as mean + SEM and statistical significance # was set at P < 0.05.

Fig. 4.

T5342126 reduced activation of microglia in both control and ethanol-dependent mice. We used immunohistochemistry to determine changes in the expression of Iba-1 (a marker of microglia) in the CeA and DG. The mice were perfused 8 days after the last T5342126 injection. We determined optical density and counted microglia-like cells in the CeA and DG of the non-dependent (control) and ethanol-dependent (CIE) mice injected with vehicle or T5342126 (control-vehicle: n = 4; control-T5342126: n = 4; vapor-vehicle: n = 3; vapor-T5342126: n = 4). (A,B) Representative staining of Iba-1 in the CeA and DG, respectively. (C) In the CeA, we found significant main effects of the ethanol-vapor exposure (F_(1,120) = 12.41, **P = 0.0006) and the T5342126 injections (F_(1,120) = 15.65, ^###P = 0.0001) on the overall abundance of Iba-1. There was no significant interaction between these factors. (D) In the DG, we did not observe significant alterations of Iba-1 abundance among the treatment groups (two-way ANOVA, P > 0.05). (E) We found significant main effects of the CIE (F_(1,120) = 4.36, *P = 0.039) and T5342126 injections (F_(1,120) = 12.52, ^##P = 0.0006) on the number of the microglia-like cells in the CeA with no significant interactions between these factors (P > 0.05). (F) In the DG, neither chronic-vapor exposure nor T5342126 injections had significant main effects on the number of microglia-like cells (two-way ANOVA, P > 0.05). (G,H) We also calculated the abundance of Iba-1 per microglia-like cell. Both the chronic-vapor exposure (F_(1,120) = 18.76, ***P < 0.0001) and T5342126 injections (F_(1,120) = 29.96, ^###P < 0.0001) had significant main effects on the Iba-1 abundance in a single microglia-like cell in the CeA, which were associated with the significant interaction (F_(1,120) = 5.54, P = 0.0202) between these factors (G). Bonferroni post-hoc test showed significant (^^^P < 0.001) increase in the Iba-1 abundance in the microglia of the chronic ethanol-vapor exposure (CIE) mice injected with vehicle compared to vehicle-injected control mice but not between the T5342126-injected CIE and control mice (P > 0.05). (H) In the DG, we found significant reduction in the abundance of Iba-1 per microglia-like cell induced by T5342126 injections in both control and vapor-exposed mice (F_(1,137) = 9.37, ^##P = 0.0027). There were neither significant main effects of the chronic ethanol-vapor exposure nor interaction between the T5342126 injections and chronic-ethanol exposure. The data are presented as mean + SEM.

Fig. 5.

The dose-dependent effects of T5342126 on anxiety and activity in the ethanol-naïve mice. The activity and anxiety were examined by the open field test. The anxiety behavior was measured by the time spent in the center of the open field and by the number of crosses the lines: (A) None of the T5342126 doses altered significantly the time spent in the center of the testing field (one-way ANOVA, F_(4,27) = 0.352, P = 0.840). (B) Except the lowest T5342126 dose (14.25 mg/kg; P = 0.127), all the higher T5342126 doses significantly reduced the number of line crosses by the naïve mice (F_(4,27) = 15.551, P < 0.0001; Fisher PLSD **P < 0.01). The effects of T5342126 on activity of the ethanol naive mice were assessed by changes in rearing counts and grooming time: (C) The higher doses of T5342126 (28.5–57 mg/kg) significantly reduced activity measured by rearing counts (F_(4,27) = 10.277, P < 0.0001; Fisher PLSD **P < 0.01). (D) T5342126 had no significant effects on the grooming of the naïve mice (F_(4,27) = 0.969, P = 0.4409). The data are presented as mean + SEM. (E) The ethanol-naïve mice drank less saccharin after a single T5342126 injection at the doses 28.5–57 mg/kg (F_(4,27) = 6.770, P < 0.0007; Fisher PLSD **P < 0.01). T5342126 decreased significantly water intake (F_(4,27) = 2.776, P = 0.0472) but only at the highest T5342126 dose (57 mg/kg; Tukey post-hoc test ^P < 0.05). (F) While there were no significant differences in baseline body temperature among the naïve mice (F_(4,27) = 0.442, P = 0.777), the T5342126 injections significantly reduced body temperature at 42.75 and 57 mg/kg (F_(4,27) = 13.977, P < 0.0001; Fisher PLSD ***P < 0.0001).