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Central administration of lipopolysaccharide induces depressive-like behavior in vivo and activates brain indoleamine 2,3 dioxygenase in murine organotypic hippocampal slice cultures.

Fu X, Zunich SM, O'Connor JC, Kavelaars A, Dantzer R, Kelley KW - J Neuroinflammation (2010)

Bottom Line: In accordance with the in vivo data, addition of LPS (10 ng/ml) to the medium of OHSCs induced steady-state expression of mRNA transcripts for IDO that peaked at 6 h and translated into increased IDO enzymatic activity within 8 h post-LPS.This activation of IDO by direct application of LPS was preceded by synthesis and secretion of TNFalpha and IL-6 protein and activation of iNOS while IFN gamma expression was undetectable.Targeting IDO itself may provide a novel therapy for inflammation-associated depression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Integrative Immunology and Behavior Program, Department of Animal Sciences, College of ACES, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT

Background: Transient stimulation of the innate immune system by an intraperitoneal injection of lipopolysaccharide (LPS) activates peripheral and central expression of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO) which mediates depressive-like behavior. It is unknown whether direct activation of the brain with LPS is sufficient to activate IDO and induce depressive-like behavior.

Methods: Sickness and depressive-like behavior in C57BL/6J mice were assessed by social exploration and the forced swim test, respectively. Expression of cytokines and IDO mRNA was measured by real-time RT-PCR and cytokine protein was measured by enzyme-linked immunosorbent assays (ELISAs). Enzymatic activity of IDO was estimated as the amount of kynurenine produced from tryptophan as determined by high pressure liquid chromatography (HPLC) with electrochemical detection.

Results: Intracerebroventricular (i.c.v.) administration of LPS (100 ng) increased steady-state transcripts of TNFalpha, IL-6 and the inducible isoform of nitric oxide synthase (iNOS) in the hippocampus in the absence of any change in IFN gamma mRNA. LPS also increased IDO expression and induced depressive-like behavior, as measured by increased duration of immobility in the forced swim test. The regulation of IDO expression was investigated using in situ organotypic hippocampal slice cultures (OHSCs) derived from brains of newborn C57BL/6J mice. In accordance with the in vivo data, addition of LPS (10 ng/ml) to the medium of OHSCs induced steady-state expression of mRNA transcripts for IDO that peaked at 6 h and translated into increased IDO enzymatic activity within 8 h post-LPS. This activation of IDO by direct application of LPS was preceded by synthesis and secretion of TNFalpha and IL-6 protein and activation of iNOS while IFN gamma expression was undetectable.

Conclusion: These data establish that activation of the innate immune system in the brain is sufficient to activate IDO and induce depressive-like behavior in the absence of detectable IFN gamma. Targeting IDO itself may provide a novel therapy for inflammation-associated depression.

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LPS induces proinflammatory cytokines and iNOS expression in OHSCs in a dose- and time-dependent manner. (A) 1, 10 or 100 ng/ml LPS were added to the medium after 10 days in culture. Tissue and media were collected 6 h later. Average Ct values for 1, 10 or 100 ng/ml LPS were, respectively, for TNFα: 20.6 ± 0.6, 19.6 ± 0.5, 19.6 ± 0.6; IL-6: 24.1 ± 0.6, 22.5 ± 0.6, 22.1 ± 0.6; iNOS: 26.2 ± 0.9, 24.9 ± 1.0, 23.6 ± 0.9. (B) Hippocampal slices were treated with LPS (10 ng/ml) and tissue and media were collected at various times. Average Ct values at 6, 12, 18 and 24 h were, respectively, for TNFα: 19.7 ± 0.1, 22.8 ± 0.7, 24.4 ± 0.9, 25.3 ± 0.7; IL-6: 23.2 ± 0.3, 24.9 ± 0.7, 28.1 ± 1.1, 28.8 ± 0.7; iNOS: 24.1 ± 0.1, 22.7 ± 0.6, 24.4 ± 1.1, 25.2 ± 1.0. Amount of the proinflammatory cytokines was measured by ELISA. Bars represent the mean ± SEM (n = 3 in each group). Bars labeled with different letters (a, b, c, d or e) are significantly different from each other at p < 0.05.
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Figure 5: LPS induces proinflammatory cytokines and iNOS expression in OHSCs in a dose- and time-dependent manner. (A) 1, 10 or 100 ng/ml LPS were added to the medium after 10 days in culture. Tissue and media were collected 6 h later. Average Ct values for 1, 10 or 100 ng/ml LPS were, respectively, for TNFα: 20.6 ± 0.6, 19.6 ± 0.5, 19.6 ± 0.6; IL-6: 24.1 ± 0.6, 22.5 ± 0.6, 22.1 ± 0.6; iNOS: 26.2 ± 0.9, 24.9 ± 1.0, 23.6 ± 0.9. (B) Hippocampal slices were treated with LPS (10 ng/ml) and tissue and media were collected at various times. Average Ct values at 6, 12, 18 and 24 h were, respectively, for TNFα: 19.7 ± 0.1, 22.8 ± 0.7, 24.4 ± 0.9, 25.3 ± 0.7; IL-6: 23.2 ± 0.3, 24.9 ± 0.7, 28.1 ± 1.1, 28.8 ± 0.7; iNOS: 24.1 ± 0.1, 22.7 ± 0.6, 24.4 ± 1.1, 25.2 ± 1.0. Amount of the proinflammatory cytokines was measured by ELISA. Bars represent the mean ± SEM (n = 3 in each group). Bars labeled with different letters (a, b, c, d or e) are significantly different from each other at p < 0.05.

Mentions: Based on the in vivo response to central injection of LPS (Fig. 2), the time point of 6 h was selected for carrying out dose-response experiments to determine the effect of LPS on cytokine expression. Slices were exposed to 1, 10 and 100 ng/ml of LPS on day 10 in culture. As shown in Fig. 5A, LPS increased TNFα and IL-6 production at both the mRNA (p < 0.05) and protein (p < 0.01) levels in a dose-dependent manner, with a maximum at 10 ng/ml. In control cultures containing medium alone, neither TNFα nor IL-6 could be detected. iNOS mRNA reached a maximum at 100 ng/ml LPS (p < 0.01) but this was not associated with a detectable increase in nitrite levels at the 6 h time point (data not shown). Nitrite levels remain undetectable in control OHSCs through at least 13 days in culture. Following LPS stimulation, nitrites begin to rise to detectable levels only after 48 h, which allows adequate amounts to accumulate in the medium (data not shown).


Central administration of lipopolysaccharide induces depressive-like behavior in vivo and activates brain indoleamine 2,3 dioxygenase in murine organotypic hippocampal slice cultures.

Fu X, Zunich SM, O'Connor JC, Kavelaars A, Dantzer R, Kelley KW - J Neuroinflammation (2010)

LPS induces proinflammatory cytokines and iNOS expression in OHSCs in a dose- and time-dependent manner. (A) 1, 10 or 100 ng/ml LPS were added to the medium after 10 days in culture. Tissue and media were collected 6 h later. Average Ct values for 1, 10 or 100 ng/ml LPS were, respectively, for TNFα: 20.6 ± 0.6, 19.6 ± 0.5, 19.6 ± 0.6; IL-6: 24.1 ± 0.6, 22.5 ± 0.6, 22.1 ± 0.6; iNOS: 26.2 ± 0.9, 24.9 ± 1.0, 23.6 ± 0.9. (B) Hippocampal slices were treated with LPS (10 ng/ml) and tissue and media were collected at various times. Average Ct values at 6, 12, 18 and 24 h were, respectively, for TNFα: 19.7 ± 0.1, 22.8 ± 0.7, 24.4 ± 0.9, 25.3 ± 0.7; IL-6: 23.2 ± 0.3, 24.9 ± 0.7, 28.1 ± 1.1, 28.8 ± 0.7; iNOS: 24.1 ± 0.1, 22.7 ± 0.6, 24.4 ± 1.1, 25.2 ± 1.0. Amount of the proinflammatory cytokines was measured by ELISA. Bars represent the mean ± SEM (n = 3 in each group). Bars labeled with different letters (a, b, c, d or e) are significantly different from each other at p < 0.05.
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Figure 5: LPS induces proinflammatory cytokines and iNOS expression in OHSCs in a dose- and time-dependent manner. (A) 1, 10 or 100 ng/ml LPS were added to the medium after 10 days in culture. Tissue and media were collected 6 h later. Average Ct values for 1, 10 or 100 ng/ml LPS were, respectively, for TNFα: 20.6 ± 0.6, 19.6 ± 0.5, 19.6 ± 0.6; IL-6: 24.1 ± 0.6, 22.5 ± 0.6, 22.1 ± 0.6; iNOS: 26.2 ± 0.9, 24.9 ± 1.0, 23.6 ± 0.9. (B) Hippocampal slices were treated with LPS (10 ng/ml) and tissue and media were collected at various times. Average Ct values at 6, 12, 18 and 24 h were, respectively, for TNFα: 19.7 ± 0.1, 22.8 ± 0.7, 24.4 ± 0.9, 25.3 ± 0.7; IL-6: 23.2 ± 0.3, 24.9 ± 0.7, 28.1 ± 1.1, 28.8 ± 0.7; iNOS: 24.1 ± 0.1, 22.7 ± 0.6, 24.4 ± 1.1, 25.2 ± 1.0. Amount of the proinflammatory cytokines was measured by ELISA. Bars represent the mean ± SEM (n = 3 in each group). Bars labeled with different letters (a, b, c, d or e) are significantly different from each other at p < 0.05.
Mentions: Based on the in vivo response to central injection of LPS (Fig. 2), the time point of 6 h was selected for carrying out dose-response experiments to determine the effect of LPS on cytokine expression. Slices were exposed to 1, 10 and 100 ng/ml of LPS on day 10 in culture. As shown in Fig. 5A, LPS increased TNFα and IL-6 production at both the mRNA (p < 0.05) and protein (p < 0.01) levels in a dose-dependent manner, with a maximum at 10 ng/ml. In control cultures containing medium alone, neither TNFα nor IL-6 could be detected. iNOS mRNA reached a maximum at 100 ng/ml LPS (p < 0.01) but this was not associated with a detectable increase in nitrite levels at the 6 h time point (data not shown). Nitrite levels remain undetectable in control OHSCs through at least 13 days in culture. Following LPS stimulation, nitrites begin to rise to detectable levels only after 48 h, which allows adequate amounts to accumulate in the medium (data not shown).

Bottom Line: In accordance with the in vivo data, addition of LPS (10 ng/ml) to the medium of OHSCs induced steady-state expression of mRNA transcripts for IDO that peaked at 6 h and translated into increased IDO enzymatic activity within 8 h post-LPS.This activation of IDO by direct application of LPS was preceded by synthesis and secretion of TNFalpha and IL-6 protein and activation of iNOS while IFN gamma expression was undetectable.Targeting IDO itself may provide a novel therapy for inflammation-associated depression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Integrative Immunology and Behavior Program, Department of Animal Sciences, College of ACES, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT

Background: Transient stimulation of the innate immune system by an intraperitoneal injection of lipopolysaccharide (LPS) activates peripheral and central expression of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO) which mediates depressive-like behavior. It is unknown whether direct activation of the brain with LPS is sufficient to activate IDO and induce depressive-like behavior.

Methods: Sickness and depressive-like behavior in C57BL/6J mice were assessed by social exploration and the forced swim test, respectively. Expression of cytokines and IDO mRNA was measured by real-time RT-PCR and cytokine protein was measured by enzyme-linked immunosorbent assays (ELISAs). Enzymatic activity of IDO was estimated as the amount of kynurenine produced from tryptophan as determined by high pressure liquid chromatography (HPLC) with electrochemical detection.

Results: Intracerebroventricular (i.c.v.) administration of LPS (100 ng) increased steady-state transcripts of TNFalpha, IL-6 and the inducible isoform of nitric oxide synthase (iNOS) in the hippocampus in the absence of any change in IFN gamma mRNA. LPS also increased IDO expression and induced depressive-like behavior, as measured by increased duration of immobility in the forced swim test. The regulation of IDO expression was investigated using in situ organotypic hippocampal slice cultures (OHSCs) derived from brains of newborn C57BL/6J mice. In accordance with the in vivo data, addition of LPS (10 ng/ml) to the medium of OHSCs induced steady-state expression of mRNA transcripts for IDO that peaked at 6 h and translated into increased IDO enzymatic activity within 8 h post-LPS. This activation of IDO by direct application of LPS was preceded by synthesis and secretion of TNFalpha and IL-6 protein and activation of iNOS while IFN gamma expression was undetectable.

Conclusion: These data establish that activation of the innate immune system in the brain is sufficient to activate IDO and induce depressive-like behavior in the absence of detectable IFN gamma. Targeting IDO itself may provide a novel therapy for inflammation-associated depression.

Show MeSH
Related in: MedlinePlus