Lipopolysaccharide-induced interleukin-6 production is controlled by glycogen synthase kinase-3 and STAT3 in the brain

Abstract

Background: Septic shock is a prevalent condition that, when not lethal, often causes disturbances in cognition, mood, and behavior, particularly due to central actions of the inflammatory cytokine interleukin-6 (IL-6). To identify potential targets to control brain IL-6, we tested if IL-6 produced by glia is regulated by signal transducer and activator of transcription-3 (STAT3) and glycogen synthase kinase-3 (GSK3).

Methods: Lipopolysaccharide (LPS) was used to induce inflammatory responses in mice or cultured primary glia. IL-6 was measured by ELISA and other inflammatory molecules were measured using an array.

Results: Mouse brain IL-6 levels increased after central, as well as peripheral, LPS administration, consistent with glia producing a portion of brain IL-6. STAT3 in the brain was activated after peripheral or central LPS administration, and in LPS-stimulated cultured primary glia. Inhibition of STAT3 expression, function, or activation reduced by ~80% IL-6 production by primary glia, demonstrating the dependence on active STAT3. GSK3 promotes STAT3 activation, and array analysis of inflammatory molecules produced by LPS-stimulated primary glia demonstrated that IL-6 was the cytokine most diminished (>90%) by GSK3 inhibition. Inhibition of GSK3, and knockdown of GSK3beta, not GSK3alpha, greatly inhibited IL-6 production by LPS-stimulated primary glia. Conversely, expression of active STAT3 and active GSK3 promoted IL-6 production. In vivo inhibition of GSK3 reduced serum and brain IL-6 levels, brain STAT3 activation, and GFAP upregulation following LPS administration.

Conclusion: STAT3 and GSK3 cooperatively promote neuroinflammation, providing novel targets for anti-inflammatory intervention.

Figure 1

Levels of cytokines in plasma and the brain parenchyma in response to sepsis. Serum and brain region levels of A, IL-6, B, TNFα, and C, IL-1β were measured 4 h after ip injection of 10 μg/g LPS or saline to evaluate the early outcomes of acute sepsis. Means ± SEM; n = 3;* p < 0.05.

Figure 2

Brain cytokines and STAT3 activation. A, IL-6, B, TNFα, C, IL-1β, D, IFNγ, and E, IL-10, were measured in homogenates (100 μg) of the ventricular infusion zone (V) or the distant occipital cortex (O), 2, 4 or 8 h after LPS (10 μg/1 μL; icv) or 4 h after saline (icv) injection. Control mice were not subjected to surgery. Means ± SEM.; n = 4–5 mice/group, *p < 0.05 4 hr treatments compared to mice injected with saline. Activation of STAT3 was measured by immunoblotting phospho-Tyr705-STAT3 and total STAT3, F, in cerebral cortex 4 h after ip injection of LPS or, G, in the ventricular infusion zone (V) or occipital cortex (O) 2, 4 or 8 h after icv infusion of LPS or 4 h after saline and compared to mice subjected to anesthesia only. STAT3 immunoblotted from cultured astrocytes is shown as a positive control.

Figure 3

STAT3 activation and cytokine production in cultured cells. A, Phospho-Tyr705-STAT3 and total STAT3 were immunoblotted after treatment with LPS (100 ng/mL) for 6 h of primary cultures of astrocytes, microglia, or neurons. Some of the neuronal cultures also were treated for 6 h with conditioned media (CM) from LPS-treated (6 h) astrocytes. B, Primary enriched astrocytes were treated with vehicle (0), or with 100 ng/mL LPS, 1 ng/mL IFNγ, or both, for 2, 4, 6, or 8 h, followed by measurements of IL-6. Means ± SEM; n = 5; *p < 0.05 compared to LPS alone. C, Primary enriched astrocytes were stimulated with LPS (100 ng/mL) and IFNγ (1 ng/mL), with or without the selective GSK3 inhibitor lithium (LiCl, 20 mM), for 6 h. Culture supernatants were incubated with an array membrane that detects 62 proteins in duplicate. Boxes indicate the location of the detected 13 proteins induced by LPS plus IFNγ. Positive controls are in the upper left and lower right corners (high intensity spots) and negative controls in the upper right corner (no spots).

Figure 4

STAT3 contributes to the LPS-induced IL-6 production in primary enriched astrocytes. Primary enriched astrocytes were treated with 100 ng/mL LPS, 1 ng/mL IFNγ, or both, for 6 h, followed by measurements of IL-6. A, Inhibition of IL-6 production after 36 h siRNA-mediated knockdown of STAT3. The knockdown efficiency was ensured by immunoblotting for STAT3 (inset). (n = 6,*p < 0.05 compared to LPS plus IFNγ with control siRNA SNC). B, Inhibition of LPS plus IFNγ (6 h) stimulated IL-6 production by inhibition of STAT3 with 10 μM JSI-124 (n = 8, *p < 0.05 compared to LPS plus IFNγ with DMSO). C, Inhibition of LPS plus IFNγ (6 h) stimulated IL-6 production by treatment with 30 (middle bars, light shade) or 100 μM AG490 (right bars, medium shading) (n = 5, *p < 0.05 compared to LPS plus IFNγ with DMSO). Means ± SEM. D, Cell viability: Primary enriched astrocytes were treated with vehicle DMSO (left, white bars), 100 ng/mL LPS, 1 ng/mL IFNγ, or both, and with 10 μM JSI-124 or 100 μM AG490 for 6 h and viability was assessed by a MTT assay. Means ± SEM; n = 3.

Figure 5

GSK3 promotes IL-6 production. A, Quantitation of the effects of lithium treatment on inflammatory molecules produced by primary enriched astrocytes stimulated with LPS (100 ng/mL) and IFNγ (1 ng/mL) for 6 h and analyzed by cytokine array, as described in Figure 3C. B, Time-dependent production of IL-6 by primary enriched astrocytes treated with vehicle (0), LPS (100 ng/mL), IFNγ (1 ng/mL), or both, in the absence and presence of 20 mM lithium. White bars indicate that no GSK3 inhibitor was added. *p < 0.05 compared to LPS plus IFNγ. C, IL-6 production by primary enriched astrocytes (n = 7) treated with 1 ng/mL IFNγ, 100 ng/mL LPS, or both, without or with 20 mM lithium or 10 μM other GSK3 inhibitors, for 6 h. White bars indicate DMSO vehicle with no GSK3 inhibitor. *p < 0.05 compared to LPS plus IFNγ without any GSK3 inhibitor. D, Cell viability: Primary enriched astrocytes were treated with 100 ng/mL LPS, 1 ng/mL IFNγ, or both, and with 20 mM lithium, 10 μM 6-bromoindirubin-3'-oxime (BIO), 10 μM kenpaullone, 10 μM GSK3 inhibitor II, or 10 μM SB216763 for 6 h and viability was assessed by a MTT assay. White bars indicate DMSO vehicle with no GSK3 inhibitor. Means ± SEM; n = 3. E, IL-6 production by primary microglia (n = 4) treated with 1 ng/mL IFNγ, 100 ng/mL LPS, or both, without or with 20 mM lithium or 10 μM SB216763, for 6 h. White bars indicate that no GSK3 inhibitor was added. *p < 0.05 compared to LPS plus IFNγ without any GSK3 inhibitor. F, IL-6 production by primary enriched astrocytes after 48 h siRNA-mediated knockdown of GSK3α, GSK3β, or both, and stimulation with LPS (100 ng/mL), IFNγ (1 ng/mL), or both, for 6 h (n = 5, *p < 0.05 compared to corresponding control siRNA SNC values). G, Cooperation between GSK3 and STAT3 to induce IL-6 production by primary enriched astrocytes, after 36 h expression of control GFP, S21A-GSK3α, or S9A-GSK3β, with or without expression of STAT3C (n = 3;*p < 0.05). Means ± SEM.

Figure 6

GSK3 inhibition attenuates IL-6 production and astrogliosis. Mice were fed for 3 weeks without or with lithium chow. A, Serum and brain region homogenate (100 μg protein) levels of IL-6 were measured 18 h after ip injection of LPS or saline vehicle control (C) to evaluate the relative long-term outcomes of acute sepsis (n = 4–6 mice/group, *p < 0.05, compared to mice injected with LPS without lithium). B, Activation of STAT3 was measured by immunoblotting phospho-Tyr705-STAT3 in brain regions 18 h after ip injection of LPS. Means ± SEM (n = 4–6 mice/group, *p < 0.05 compared to mice injected with LPS without lithium). C, GFAP in brain regions 18 h after LPS (ip). Means ± SEM (n = 4–6 mice/group,*p < 0.05, compared to mice injected with LPS without lithium).