Minocycline attenuates lipopolysaccharide (LPS)-induced neuroinflammation, sickness behavior, and anhedonia

Abstract

Background: Activation of the peripheral innate immune system stimulates the secretion of CNS cytokines that modulate the behavioral symptoms of sickness. Excessive production of cytokines by microglia, however, may cause long-lasting behavioral and cognitive complications. The purpose of this study was to determine if minocycline, an anti-inflammatory agent and purported microglial inhibitor, attenuates lipopolysaccharide (LPS)-induced neuroinflammation, sickness behavior, and anhedonia.

Methods: In the first set of experiments the effect of minocycline pretreatment on LPS-induced microglia activation was assessed in BV-2 microglia cell cultures. In the second study, adult (3-6 m) BALB/c mice received an intraperitoneal (i.p.) injection of vehicle or minocycline (50 mg/kg) for three consecutive days. On the third day, mice were also injected (i.p.) with saline or Escherichia coli LPS (0.33 mg/kg) and behavior (i.e., sickness and anhedonia) and markers of neuroinflammation (i.e., microglia activation and inflammatory cytokines) were determined. In the final study, adult and aged BALB/c mice were treated with the same minocycline and LPS injection regimen and markers of neuroinflammation were determined. All data were analyzed using Statistical Analysis Systems General Linear Model procedures and were subjected to one-, two-, or three-way ANOVA to determine significant main effects and interactions.

Results: Minocycline blocked LPS-stimulated inflammatory cytokine secretion in the BV-2 microglia-derived cell line and reduced LPS-induced Toll-like-receptor-2 (TLR2) surface expression on brain microglia. Moreover, minocycline facilitated the recovery from sickness behavior (i.e., anorexia, weight loss, and social withdrawal) and prevented anhedonia in adult mice challenged with LPS. Furthermore, the minocycline associated recovery from LPS-induced sickness behavior was paralleled by reduced mRNA levels of Interleukin (IL)-1beta, IL-6, and indoleamine 2, 3 dioxygenase (IDO) in the cortex and hippocampus. Finally, in aged mice, where exaggerated neuroinflammation was elicited by LPS, minocycline pretreatment was still effective in markedly reducing mRNA levels of IL-1beta, TLR2 and IDO in the hippocampus.

Conclusion: These data indicate that minocycline mitigates neuroinflammation in the adult and aged brain and modulates the cytokine-associated changes in motivation and behavior.

Figure 1

Minocycline suppresses LPS-stimulated expression and production of cytokines in BV-2 microglia cultures. A) BV-2 cells were stimulated with 0, 0.1, 1, 10, 100, or 1000 ng/ml LPS and IL-6 protein levels were determined from supernatants collected 4 h later. B) BV-2 cells were pretreated with 0, 25, 50, 100, 200, or 400 μg/ml minocycline for 30 min and then treated with LPS (10 ng/ml). IL-6 protein levels were determined from supernatants collected 4 h later. C) BV-2 cells were treated with 200 μg/ml minocycline for 30 min and then treated with 10 ng/ml LPS. IL-1β protein levels were determined from supernatants collected 4 h later. D) BV-2 cells were treated as above and MHC II, TLR2, IL-1β, and IL-6 mRNA levels were determined. For each cell culture experiment, results are an average of 4 independent experiments. Means with different letters (a, b, c, d, or e) are significantly different (P < 0.05) from each other.

Figure 2

LPS-induced TLR2 surface expression on microglia is reduced by minocycline. Adult mice were injected i.p. with vehicle or minocycline for three consecutive days. On the third day mice were challenged with saline or LPS i.p., microglia and macrophages were collected 24 h later and TLR2 surface expression was determined. A) Representative bivariate density plot of stained cells. Macrophages were CD11b⁺/CD45^high and microglia were CD11b⁺/CD45^low. B&C) Representative histograms of TLR2 expression on microglia following experimental treatments. C) Mean fluorescence intensity (M.F.I.) of TLR2 expression on microglia (CD11b⁺/CD45^low) following experimental treatments. Bars represent the mean ± SEM (n = 6). Means with different letters (a, b, or c) are significantly different (P < 0.05) from each other.

Figure 3

Minocycline facilitates recovery from LPS-induced sickness behavior. Mice were injected i.p. with vehicle or minocycline for three consecutive days. On the third day mice were challenged with saline or LPS i.p. A) Social exploratory behavior was measured before i.p. LPS injection and again 2, 4, 8, and 24 h later. Graph represents the mean ± SEM (n = 10). Means with * are significantly different (P < 0.05) from saline controls and means with ‡ are significantly different from Vehicle LPS. In the same experiment, B) body weight and C) food intake were measured before i.p. LPS injection and again 2, 4, 8, and 24 h later. Bars represent the mean ± SEM (n = 10). Means with different letters (a, b, c, or d) are significantly different (P < 0.05) from each other.

Figure 4

LPS-associated anhedonia is blocked by minocycline. Mice were injected i.p. with vehicle or minocycline for three consecutive days. On the third day mice were challenged with saline or LPS i.p. and sucrose preference was determined 24 to 39 h post LPS. Bars represent the mean ± SEM (n = 15). Means with different letters (a or b) are significantly different (P < 0.05) from each other.

Figure 5

Minocycline attenuates LPS-induced mRNA levels of IL-1β, IL-6, and IDO in the cortex and hippocampus of adult mice. Mice were injected i.p. with vehicle or minocycline for three consecutive days. On the third day mice were challenged with either saline or LPS i.p and A) IL-1β, B) IL-6, and C) IDO mRNA levels were determined in the cortex and hippocampus collected 4 h later. Bars represent the mean ± SEM (n = 8). For each brain region, means with different letters (a, b, or c) are significantly different (P < 0.05) from each other.

Figure 6

Minocycline reduces LPS-induced IL-6, but not IL-1β, levels in the plasma. Mice were injected i.p. with vehicle or minocycline for three consecutive days. On the third day mice were injected with saline or LPS i.p. IL-1β and IL-6 were determined in plasma collected 4 h later. There was no detectable IL-6 (n.d.) in the plasma of saline-treated mice. Bars represent the mean ± SEM (n = 8). Means with different letters (a, b, or c) are significantly different (P < 0.05) from each other.

Figure 7

Minocycline pretreatment attenuates LPS-induced mRNA levels of TLR2, IL-1β, IL-6, and IDO in the hippocampus of aged mice. Adult and aged mice were injected i.p. with vehicle or minocycline for three consecutive days. On the third day mice were challenged with saline or LPS i.p. and A) MHC II, B) TLR2, C) IL-1β, D) IL-6, and E) IDO mRNA levels were determined from hippocampus collected 4 h later. Bars represent the mean ± SEM (n = 8). Means with different letters (a, b, c, or d) are significantly different (P < 0.05) from each other.