IL-1beta is an essential mediator of the antineurogenic and anhedonic effects of stress

Abstract

Stress decreases neurogenesis in the adult hippocampus, and blockade of this effect is required for the actions of antidepressants in behavioral models of depression. However, the mechanisms underlying these effects of stress have not been identified. Here, we demonstrate an essential role for the proinflammatory cytokine IL-1beta. Administration of IL-1beta or acute stress suppressed hippocampal cell proliferation. Blockade of the IL-1beta receptor, IL-1RI, by using either an inhibitor or IL-1RI null mice blocks the antineurogenic effect of stress and blocks the anhedonic behavior caused by chronic stress exposure. In vivo and in vitro studies demonstrate that hippocampal neural progenitor cells express IL-1RI and that activation of this receptor decreases cell proliferation via the nuclear factor-kappaB signaling pathway. These findings demonstrate that IL-1beta is a critical mediator of the antineurogenic and depressive-like behavior caused by acute and chronic stress.

Fig. 1.

Effects of IL-1β, acute stress, and/or IL-1Ra on cell proliferation and cell death in the DG of the hippocampus in rats. (A) IL-1β administration (20 or 100 ng per rat) decreased the number of BrdU⁺ cells in the DG (F_2,26 = 4.059, P < 0.05; n = 8–9 per group). (B) IL-1Ra administration (0.25 μg per rat) blocked the antiproliferative effect of IL-1β (100 ng per rat) (F_3,36 = 5.205, P < 0.01; n = 7–10 per group). (C) IL-1β did not change the number of TUNEL⁺ cells per DG (t₆ = 0.674, P = n.s.; n = 4 per group). (D and E) Dividing BrdU⁺ cells (black arrowheads) in the DG of immobilization (IMM)-stressed (D) and IL-1Ra infused IMM-stressed rats (E). (Scale bar, 100 μm.) (F) A representative TUNEL⁺ cell (red) in the DG. Granular cell layer (GCL) is shown with DAPI+ staining (blue). SGZ, subgranular zone, (Scale bar, 20 μm.) (G and H) Inescapable foot-shock (FS) (G) and IMM stress (H) exposures also decreased the number of BrdU⁺ cells in the DG. The antiproliferative effect of acute stress was blocked by IL-1Ra (0.25 μg per rat) (FS, F_3,30 = 6.298, P < 0.01; IMM, F_3,34 = 5,445, P < 0.01; n = 5–9 per group). (I) IMM did not affect the number of TUNEL⁺ cells per DG (t₆ = 1.450, P = n.s.; n = 4 per group). (J) A schematic diagram depicting the experimental procedures for acute stress. After 10-day recovery and handling, followed by cannulation, animals were infused with IL-1Ra or vehicle immediately before stress and given BrdU immediately after stress. Rats were killed (sac) 2 h after BrdU administration. *, P < 0.05 compared with CTR; #, P < 0.05 compared with 100 ng of IL-1β-infused or stressed rats in the Tukey's post hoc test, mean ± SEM.

Fig. 2.

Effects of IL-1Ra during chronic unpredictable stress (CUS) on sucrose preference and hippocampal neurogenesis in rats. (A) A schematic diagram depicting the experimental procedures for CUS in rats. After 5-day recovery, preceded by implantation of cannula (i.c.v.) and minipump (s.c.), all rats were exposed to two stressors per day for 21 days and received BrdU daily for 4 days of the last CUS period. Immediately before (PRE-CUS) and after CUS (POST-CUS), sucrose preference test (SPT) was conducted. Rats were killed 14 days after last BrdU administration. (B) Continuous administration of IL-1Ra during CUS restored the sucrose preference that was reduced by CUS (main effect treatment: F_1,27 = 11.927, P < 0.01; main effect time: F_1,27 = 8.675, P < 0.01; treatment × time: F_1,27 = 7.765, P < 0.05; n = 7 per group). **, P < 0.01 compared with CUS + 1Ra group; ##, P < 0.01 compared with Pre-PBS group Tukey's post hoc test. (C–E) There was a significant difference in the number of hippocampal BrdU⁺ cells in CUS + 1Ra rats (D) relative to CUS+PBS rats (C) (t₁₂ = 2.333, P < 0.05; n = 7 per group). (Scale bar, 100 μm.) (F and G) IL-1Ra during CUS also increased the number of new immature neurons measured as the number of BrdU⁺ (red) and DCX⁺ (green) double-labeled cells per hippocampal DG (t₆ = 3.431, P < 0.05; n = 4 per group). *, P < 0.05 in the t test. mean ± SEM. (Scale bar, 25 μm.)

Fig. 3.

Effects of acute and chronic stress on hippocampal neurogenesis (cell proliferation) and sucrose consumption in IL-1RI null mice. (A–C) Acute stress (aSTR, immobilization and rotation, 50 min) decreased the number of BrdU⁺ cells in the DG of WT (A) but not IL-1RI null mice (KO, B) (main effect aSTR: F_1,31 = 8.297, P < 0.01; main effect genotype: F_1,31 = 6.874, P < 0.05; aSTR × genotype: F_1,31 = 1.559, P = n.s.; n = 8 per group). Note that the number of BrdU⁺ cells was not significantly different between nonstressed (Non-aSTR) WT and KO mice, indicating no difference in the baseline effect of IL-1RI null mutation. (D) Four-week CUS also significantly decreased the BrdU⁺ cell numbers in WT mice but not in IL-1RI KO mice (F_1,22 = 1.933, P = n.s.; n = 5–6 per group). CUS KO mice showed more BrdU⁺ cells than CUS WT mice. There was also no difference in baseline. (E) CUS also decreased the number of immature neurons, determined by colabeling of BrdU with DCX, and this effect was blocked in the IL-1RI KO mice. (F) In the sucrose consumption test, there was no baseline difference between WT and IL-1RI KO mice. CUS significantly decreased sucrose consumption in WT but not in IL-1RI KO mice (main effect CUS: F_1,22 = 1.361, P = n.s.; main effect genotype: F_1,22 = 5.191, P < 0.05; CUS × genotype: F_1,22 = 11.388, P < 0.01). (G) There was no difference in water consumption among groups (main effect CUS: F_1,22 = 0.305, P = n.s.; main effect genotype: F_1,22 = 0.040, P = n.s.; CUS × genotype: F_1,22 = 0.006, P = n.s.). (H) Experimental procedures for CUS in mice. CUS mice were exposed to two or three stressors per day for 28 days and received BrdU daily for 4 days of the last CUS period. On the last CUS day, sucrose consumption test (SCT) was conducted. Mice were killed 14 days after last BrdU administration. **, P < 0.01 compared with nonstressed WT mice; #, P < 0.05, ##, P < 0.01 compared with stressed WT mice in the Tukey's post hoc test, mean ± SEM.

Fig. 4.

Expression of IL-1RI in the hippocampus in rats. IL-1RI (green) is expressed in proliferating cells (BrdU+, red, A–C) and neural progenitors (D–F, sox²⁺, red) in the SGZ of the hippocampus. (Scale bars, 50 μm.)

Fig. 5.

Expression of IL-1RI in primary cultured AHPs and effects of IL-1β and/or IL-1Ra on AHP proliferation. (A) All of the AHPs (nestin⁺, green) in vitro coexpressed IL-1RI (red). (B) Proliferating AHPs (BrdU⁺, red) also expressed IL-1RI (green). (C) Expression of IL-1RI genes in cultured AHPs. RT-PCR was performed with IL-1RI and GAPDH genes prepared from cultured AHPs in the presence (left lane) or absence (right lane, as a negative control) of reverse transcriptase (RT). (D and E) Representative images of proliferating (BrdU⁺, red) AHPs (nestin⁺, green) in the presence of PBS (D) or IL-1β (E, 10 ng/ml). (F and G) IL-1β did not alter the percentage of TUNEL⁺ (red) to DAPI⁺ (blue) cells (n = 4) (t₆ = 0.194, P = n.s.; n = 4 per group). (H) IL-1Ra (100 ng/ml) blocked the IL-1β (10 ng/ml)-dependent decrease of cyclin D1 expression (F_3,15 = 7.329, P < 0.01; n = 3–4 per group). β-Actin served as an internal loading control. Expression levels were measured with relative optical density (OD). (I) IL-1β (10–100 ng/ml) decreased the ratio of BrdU⁺ (red) to nestin⁺ (green) compared with CTR (0 ng/ml IL-1β) (F_3,33 = 7.156, P < 0.001; n = 8–9 per group). (J) Coincubation of IL-1Ra (100 ng/ml) blocked the antiproliferative effect of IL-1β (F_6,39 = 4.198, P < 0.01; n = 5–6 per group). (K) Coincubation of NF-κB (JSH-23, 25 μM) or Iκκ inhibitors (SC-514, 25 μM) blocked the antiproliferative effect of IL-1β (F_5,35 = 8.504, P < 0.001; n = 5–6 per group). (Scale bars, 50 μm.) *, P < 0.05 and **, P < 0.01 compared with CTR. ##, P < 0.01 and ###, P < 0.001 compared with 10 ng/ml IL-1β group in the Tukey's post hoc tests, mean ± SEM.