Genes involved in the balance between neuronal survival and death during inflammation - PubMed (original) (raw)

Genes involved in the balance between neuronal survival and death during inflammation

Isaias Glezer et al. PLoS One. 2007.

Abstract

Glucocorticoids are potent regulators of the innate immune response, and alteration in this inhibitory feedback has detrimental consequences for the neural tissue. This study profiled and investigated functionally candidate genes mediating this switch between cell survival and death during an acute inflammatory reaction subsequent to the absence of glucocorticoid signaling. Oligonucleotide microarray analysis revealed that following lipopolysaccharide (LPS) intracerebral administration at striatum level, more modulated genes presented transcription impairment than exacerbation upon glucocorticoid receptor blockage. Among impaired genes we identified ceruloplasmin (Cp), which plays a key role in iron metabolism and is implicated in a neurodegenative disease. Microglial and endothelial induction of Cp is a natural neuroprotective mechanism during inflammation, because Cp-deficient mice exhibited increased iron accumulation and demyelination when exposed to LPS and neurovascular reactivity to pneumococcal meningitis. This study has identified genes that can play a critical role in programming the innate immune response, helping to clarify the mechanisms leading to protection or damage during inflammatory conditions in the CNS.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1

Gene profiling of CNS LPS response in presence or absence of functional GR signaling. (A) Treatment schedule of animals. (B and C) Schematic representation of the injection site and brain area dissected for RNA isolation, respectively. (D) A 2-contrast plot of genes selected as differently expressed in ipsilateral side when LPS injection was performed in mice pre-treated or not with RU486. The abscissa axis represents the difference (contrast) between LPS/RU486 treatment and LPS single treatment (RMA expression levels); ordinate axis represents the LPS vs. Saline contrast. Red dots represent ipsilateral contrast value; green dots, represent contralateral contrast values. Genes highlighted in the diagram were selected for validation experiments. Dashed lines: 30% fold-change. (E) Similar to “D”, but in regard to contralateral side. (F) Representation of the gene expression behavior associated with RU486 effects on LPS-induced changes, defining how these groups of genes are described in the text. Abbreviations: AP, antero-posterior; contra, contralateral; DV, dorso-ventral; i.p., intra-peritoneal; ipsi, ipsilateral; i.s., intrastriatal; L, lateral.

Figure 2

Figure 2

Functional classification and biological relevance of the differently expressed genes. The plot showing significant biological processes (hypergeometric distribution) associated with six different lists of differently expressed genes (LPS upregulated, LPS upregulation exacerbated by RU486, LPS upregulation prevented by RU486, LPS downregulated, LPS dowregulation prevented by RU486 and RU486 main effect). Hierarchical clustering of the Gene Ontology nodes was performed as described in text S1. A color/intensity code assigns number of genes and p-value for each biological process associated with the lists' heat map.

Figure 3

Figure 3

Bimodal RU486 effect on CNS inflammation as demonstrated by APRs and genes only responsive to LPS upon GR-antagonism. (A) Dark-field photomicrographs showing hybridization signals of the specified transcripts from mice that received saline or, LPS or, LPS/RU486 combined treatments. (B) Semi-quantitative analysis of mRNA levels [optical density (O.D.)] in mouse brains 12 h after the intracerebral infusions. Absence (-) or presence (+) of LPS or increasing doses of RU486 is indicated. Quantification methods may vary among transcripts; see “Experimental Procedures” section. Results represent means ± SEM of 3–5 mice per group. One-way ANOVA followed by a Tukey's HSD multiple comparison test: significantly different (* p<0.05, ** p<0.01, *** p<0.005) from the saline-injected group; significantly different (# p<0.05) from the LPS-treated group. Abbreviations: BnST, bed nucleus of stria terminalis; bv, blood vessel; Cx, cortex; Ipsi, ipsilateral; LV, lateral ventricle; m, meninges; MPO, medial preoptic nucleus.

Figure 4

Figure 4

Validation of genes upregulated or downregulated by LPS in a glucocorticoid-dependent manner. (A) Representative examples of coronal sections depicting LPS induced changes in gene expression of the indicated transcripts. (B) Semi-quantitative analysis of mRNA levels (O.D.) in mouse brains 12 h after the intracerebral infusions. One-way ANOVA followed by a Tukey's HSD multiple comparison test: significantly different (* p<0.05, ** p<0.01, *** p<0.005) from the saline-injected group; significantly different (# p<0.05, ### p<0.005) from the LPS-treated group. Abbreviations: Ipsi, ipsilateral; LV, lateral ventricle; Pir, piriform cortex.

Figure 5

Figure 5

Characterization of ceruloplasmin (Cp) expression following an acute innate immune challenge. (A) Hybridization signals of Cp riboprobe on brain sections of saline-, or LPS-, or LPS/RU486-treated mice, sacrificed 12 h after the intrastriatal injection. (B) Semi-quantitative analysis of Cp mRNA levels (O.D.) depicted in “A”. (C) Results of quantitative reverse-transcriptase PCR (qPCR). (D) RMA expression levels of four different probe sets to detect Cp transcript selected according to a significant LPS effect. (E) Representative microphotographs of labeling experiments combining immunohistochemistry with in situ hybridization. Silver grain signals (Cp mRNA) overlap IBA1-, GFAP- and CD31-positive cells. (F) Confocal microscopy results showing co-localization of CP protein with CD31-, IBA1- and GFAP-positive cells, but not with MAC-2-positive cells, in brain sections from animals sacrificed 24 h after LPS challenge. Dark arrowheads: co-localization of silver grains with immunolabeling brown staining. Empty white arrows: co-localization determined by confocal laser scanning. Full white arrows and white arrowheads were used to identify cell types that failed to show convincing co-localization. One-way ANOVA followed by a Tukey'S HSD multiple comparison test: significantly different (** p<0.01, *** p<0.005) from the saline-injected group; significantly different (# p<0.05, ### p<0.005) from the LPS-treated group. Abbreviations: bv, blood vessel; m, meningis.

Figure 6

Figure 6

Ceruloplasmin regulates brain iron levels during inflammation and confers neuroprotection. (A) Cp+/+, +/- or -/- mice were sacrificed 2 days after LPS infusion and brain sections stained for Iron III (Perls' staining). (B) High magnification of iron labeling (representative brain sections from _Cp_-/-mice sacrificed 2 or 5 days after LPS infusion). (C) Surface analysis to determine extent of iron deposition. (D) Fluoro-Jade B (FJB) staining was performed in brain sections from Cp+/+, +/- and -/- infused with LPS or saline. Volumetric analysis was performed by stereological procedure. (E) Representative dark-field picture of adjacent brain sections hybridized with Tlr2 cRNA probe in LPS treated mice 2 days after infusion. (F) Semi-quantitative analysis of Tlr2 mRNA hybridization signal. (G) Representative sections hybridized with Plp cRNA riboprobe to detect regions lacking signals (demyelinated area). These panels show two different coronal levels (cannulae track level and a section distal to the site of injection). (H) Surface analysis of demyelinated area. (I) and (J) are representative microphotographs of Sudam Black B (SBB) stained sections from Cp+/+ and -/- mice sacrificed 2 days after LPS infusion, as indicated. Statistical analysis was performed via a two or three-way ANOVA followed by a Bonferroni's multiple comparison test when applicable. “a”, significant genotype effect (p<0.05), significant time effect (p<0.01), significant treatment effect (p<0.001), _Cp_-/-significantly different from Cp+/+ (p<0.01) and Cp+/- (p<0.05). “b”, significant genotype effect (p<0.01), _Cp_-/- statistically different from Cp+/+ (p<0.005). “c”, significant interaction between treatment and time factors (p<0.01), LPS treatment 2 days statistically different from LPS 5 days (p<0.001). “d”, significant interaction between treatment and genotype effect (p<0.05), significant time effect (p<0.001), in the LPS treated group _Cp_-/- was statistically different from Cp+/+ († p<0.005) and from Cp+/- (‡ p<0.01). Abbreviations: cc, corpus callosum; CPu, caudate putamen; ct, cannulae track; Cx, cortex; LV, lateral ventricle.

Figure 7

Figure 7

Modulation of cerebro-vascular response by ceruloplasmin during early phases of pneumococcal infection. (A) Survival curves of Cp+/+, +/- and -/- following inoculation with S. Penumoniae (2 week follow up). (B) Ceruloplasmin protein immunoreactivity at the vascular level in the hindbrain of a Cp+/+ infected mouse; _Cp_-/- mice was used as control for the primary antibody. (C) Dark-field photomicrographs showing representative hybridization signals of Nfkbia or Selp riboprobes on hindbrain sections of Cp+/+ or -/- mice killed at the early phase of infection (36–48 h post-infection). (D) High magnification photomicrographs depicting the expression pattern of Selp mRNA associated with large blood vessels and not in cells lining the meninges or the ventricle. (E) Semi-quantitative analysis of Nfkbia and Selp mRNA levels O.D. Student's t-test was used to compare Cp+/+ and _Cp_-/- means (see text for details); * p<0.05. Abbreviations: bv, blood vessel; Cb, cerebellum; IC, inferior colliculus; m, meninges.

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