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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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Proinflammatory cytokines and iNOS expression in hippocampal slices at the mRNA and protein level. Day 0 represents freshly-isolated hippocampi that were subjected to transversal slicing and mRNA being prepared immediately following slicing. Culture media and slice tissue were collected on days 1, 3, 7, 10 and 14 after the start of the culture. LPS (10 ng/ml) was used as a positive control on day 10, with mRNA and protein being measured 6 h later. Steady-state expression of mRNA transcripts was measured by real-time RT-PCR, and proinflammatory cytokines in the media were measured by ELISA. Average Ct values for at 1, 3, 7, 10 and 14 days for each mRNA species were, respectively: TNFα: 27.6 ± 0.7, 27.8 ± 0.5, 29.0 ± 0.3, 29.1 ± 0.5, 28.7 ± 0.6; IL-6: 30.7 ± 0.4, 34.8 ± 0.2, 36.1 ± 0.9, 35.8 ± 0.8, 36.6 ± 1.1; iNOS: 28.8 ± 0.8, 31.8 ± 0.8, 31.1 ± 1.0, 32.9 ± 0.3, 35.5 ± 0.8. Data represent the mean ± SEM (n = 3 in each group). Bars labeled with different letters (a, b, c or d) are significantly different from each other at p < 0.05.
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Figure 3: Proinflammatory cytokines and iNOS expression in hippocampal slices at the mRNA and protein level. Day 0 represents freshly-isolated hippocampi that were subjected to transversal slicing and mRNA being prepared immediately following slicing. Culture media and slice tissue were collected on days 1, 3, 7, 10 and 14 after the start of the culture. LPS (10 ng/ml) was used as a positive control on day 10, with mRNA and protein being measured 6 h later. Steady-state expression of mRNA transcripts was measured by real-time RT-PCR, and proinflammatory cytokines in the media were measured by ELISA. Average Ct values for at 1, 3, 7, 10 and 14 days for each mRNA species were, respectively: TNFα: 27.6 ± 0.7, 27.8 ± 0.5, 29.0 ± 0.3, 29.1 ± 0.5, 28.7 ± 0.6; IL-6: 30.7 ± 0.4, 34.8 ± 0.2, 36.1 ± 0.9, 35.8 ± 0.8, 36.6 ± 1.1; iNOS: 28.8 ± 0.8, 31.8 ± 0.8, 31.1 ± 1.0, 32.9 ± 0.3, 35.5 ± 0.8. Data represent the mean ± SEM (n = 3 in each group). Bars labeled with different letters (a, b, c or d) are significantly different from each other at p < 0.05.

Mentions: Immediately after transversal slicing on day 0, mRNA from hippocampi was isolated. Although mRNA for TNFα, IL-6 and iNOS could be detected immediately after slicing on day 0, the amount was extremely low, particularly compared to the amount induced by LPS later in culture (Fig. 3). Indeed, after 10 days in culture, treatment with LPS dramatically increased expression of TNFα, IL-6 and iNOS mRNA and induced roughly a nanogram per milliliter secretion of TNFα and IL-6 protein in the culture medium (Fig. 3). The amount of mRNA and protein caused by transversal slicing was minimal compared to treatment with LPS, but nonetheless, was detectable in several instances. For example, TNFα mRNA increased on both days 1 and 3 (p < 0.01) after slicing, but it returned to low but detectable levels by day 7. However, the increase in amplified mRNA for TNFα was not accompanied by any detectable TNFα released into the medium at any time point. There was a temporary increase in IL-6 mRNA on day 1 (Fig. 3, p < 0.01). IL-6 protein in the culture medium was significantly increased on days 1 and 3 (p < 0.01). Expression of iNOS mRNA was only slightly increased (3-fold; p < 0.01) on day 1. This modest increase in iNOS mRNA was not associated with any increase in nitrite levels in the culture medium, as measured by Griess reaction (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)

Proinflammatory cytokines and iNOS expression in hippocampal slices at the mRNA and protein level. Day 0 represents freshly-isolated hippocampi that were subjected to transversal slicing and mRNA being prepared immediately following slicing. Culture media and slice tissue were collected on days 1, 3, 7, 10 and 14 after the start of the culture. LPS (10 ng/ml) was used as a positive control on day 10, with mRNA and protein being measured 6 h later. Steady-state expression of mRNA transcripts was measured by real-time RT-PCR, and proinflammatory cytokines in the media were measured by ELISA. Average Ct values for at 1, 3, 7, 10 and 14 days for each mRNA species were, respectively: TNFα: 27.6 ± 0.7, 27.8 ± 0.5, 29.0 ± 0.3, 29.1 ± 0.5, 28.7 ± 0.6; IL-6: 30.7 ± 0.4, 34.8 ± 0.2, 36.1 ± 0.9, 35.8 ± 0.8, 36.6 ± 1.1; iNOS: 28.8 ± 0.8, 31.8 ± 0.8, 31.1 ± 1.0, 32.9 ± 0.3, 35.5 ± 0.8. Data represent the mean ± SEM (n = 3 in each group). Bars labeled with different letters (a, b, c or d) are significantly different from each other at p < 0.05.
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Related In: Results  -  Collection

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Figure 3: Proinflammatory cytokines and iNOS expression in hippocampal slices at the mRNA and protein level. Day 0 represents freshly-isolated hippocampi that were subjected to transversal slicing and mRNA being prepared immediately following slicing. Culture media and slice tissue were collected on days 1, 3, 7, 10 and 14 after the start of the culture. LPS (10 ng/ml) was used as a positive control on day 10, with mRNA and protein being measured 6 h later. Steady-state expression of mRNA transcripts was measured by real-time RT-PCR, and proinflammatory cytokines in the media were measured by ELISA. Average Ct values for at 1, 3, 7, 10 and 14 days for each mRNA species were, respectively: TNFα: 27.6 ± 0.7, 27.8 ± 0.5, 29.0 ± 0.3, 29.1 ± 0.5, 28.7 ± 0.6; IL-6: 30.7 ± 0.4, 34.8 ± 0.2, 36.1 ± 0.9, 35.8 ± 0.8, 36.6 ± 1.1; iNOS: 28.8 ± 0.8, 31.8 ± 0.8, 31.1 ± 1.0, 32.9 ± 0.3, 35.5 ± 0.8. Data represent the mean ± SEM (n = 3 in each group). Bars labeled with different letters (a, b, c or d) are significantly different from each other at p < 0.05.
Mentions: Immediately after transversal slicing on day 0, mRNA from hippocampi was isolated. Although mRNA for TNFα, IL-6 and iNOS could be detected immediately after slicing on day 0, the amount was extremely low, particularly compared to the amount induced by LPS later in culture (Fig. 3). Indeed, after 10 days in culture, treatment with LPS dramatically increased expression of TNFα, IL-6 and iNOS mRNA and induced roughly a nanogram per milliliter secretion of TNFα and IL-6 protein in the culture medium (Fig. 3). The amount of mRNA and protein caused by transversal slicing was minimal compared to treatment with LPS, but nonetheless, was detectable in several instances. For example, TNFα mRNA increased on both days 1 and 3 (p < 0.01) after slicing, but it returned to low but detectable levels by day 7. However, the increase in amplified mRNA for TNFα was not accompanied by any detectable TNFα released into the medium at any time point. There was a temporary increase in IL-6 mRNA on day 1 (Fig. 3, p < 0.01). IL-6 protein in the culture medium was significantly increased on days 1 and 3 (p < 0.01). Expression of iNOS mRNA was only slightly increased (3-fold; p < 0.01) on day 1. This modest increase in iNOS mRNA was not associated with any increase in nitrite levels in the culture medium, as measured by Griess reaction (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