Modelling Neuroinflammation in vitro: a tool to test the potential neuroprotective effect of anti-inflammatory agents

Neuron-microglia co-cultures treated with pro-inflammatory agents are a useful tool to study neuroinflammation in vitro, where to test the potential neuroprotective effect of anti-inflammatory compounds. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. We compared the use of neuron-primary microglia and neuron-BV2 cells (a microglial cell line) co-cultures, using different neuron:microglia ratios, treatments and time post-treatment to induce glial activation and derived neurotoxicity. We show that each model requires different experimental conditions, but that both neuron-BV2 and neuron-primary microglia LPS/IFN-γ-treated co-cultures are good to study the potential neuroprotective effect of anti-inflammatory agents. The contribution of different pro-inflammatory parameters in the neurotoxicity induced by reactive microglial cells was determined. IL-10 pre-treatment completely inhibited LPS/IFN-γ-induced TNF-α and IL-6 release, and COX-2 expression both in BV2 and primary microglial cultures, but not NO production and iNOS expression. However, LPS/IFN-γ induced neurotoxicity was not inhibited in IL-10 pre-treated co-cultures. The inhibition of NO production using the specific iNOS inhibitor 1400 W totally abolished the neurotoxic effect of LPS/IFN-γ, suggesting a major role for NO in the neurotoxic effect of activated microglia. Consequently, among the anti-inflammatory agents, special attention should be paid to compounds that inhibit NO production

Pro-inflammatory gene expression and neurotoxic effects of activated microglia are attenuated by absence of CCAAT/enhancer binding protein β

Background. Microglia and astrocytes respond to homeostatic disturbances with profound changes of gene expression. This response, known as glial activation or neuroinflammation, can be detrimental to the surrounding tissue. The transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is an important regulator of gene expression in inflammation but little is known about its involvement in glial activation. To explore the functional role of C/EBPß in glial activation we have analyzed pro-inflammatory gene expression and neurotoxicity in murine wild type and C/EBPß-null glial cultures. Methods. Due to fertility and mortality problems associated with the C/EBPß-null genotype we developed a protocol to prepare mixed glial cultures from cerebral cortex of a single mouse embryo with high yield. Wild-type and C/EBPß-null glial cultures were compared in terms of total cell density by Hoechst-33258 staining; microglial content by CD11b immunocytochemistry; astroglial content by GFAP western blot; gene expression by quantitative real-time PCR, western blot, immunocytochemistry and Griess reaction; and microglial neurotoxicity by estimating MAP2 content in neuronal/microglial cocultures. C/EBPß DNA binding activity was evaluated by electrophoretic mobility shift assay and quantitative chromatin immunoprecipitation. Results. C/EBPß mRNA and protein levels, as well as DNA binding, were increased in glial cultures by treatment with lipopolysaccharide (LPS) or LPS + interferon ¿ (IFN¿). Quantitative chromatin immunoprecipitation showed binding of C/EBPß to pro-inflammatory gene promoters in glial activation in a stimulus- and gene-dependent manner. In agreement with these results, LPS and LPS+IFN¿ induced different transcriptional patterns between pro-inflammatory cytokines and NO synthase-2 genes. Furthermore, the expressions of IL-1ß and NO synthase-2, and consequent NO production, were reduced in the absence of C/EBPß. In addition, neurotoxicity elicited by LPS+IFN¿-treated microglia co-cultured with neurons was completely abolished by the absence of C/EBPß in microglia

IL-10 pre-treatment did not inhibit the neurotoxicity induced by reactive microglia in neuron-primary microglia co-cultures.

<p>Neuron-primary microglia co-cultures were treated with 100 ng/mL LPS +30 ng/mL IFN-γ for 48 h in the presence or absence of 50 ng/mL of IL-10 administered 1 h prior to LPS/IFN-γ. Evaluation of neuronal viability by MAP2-ABTS-ELISA assay. Results are presented as % of MAP2 immunostaining in control co-cultures. Bars are means + SEM of four independent experiments. **p<0.01 vs control; one-way ANOVA (repeated measures) and Newman-Keuls post-test.</p

Glial activation and resulting neurotoxicity in neuron-BV2 co-cultures.

<p>Neuronal viability (MAP2-ABTS-ELISA assay) in neuron-BV2 co-cultures 24 h or 48 h after treatment with 100 ng/mL LPS +0.5 ng/mL IFN-γ (A) or 100 ng/mL LPS +5 ng/mL IFN-γ (B). Results are presented as % of MAP2 immunostaining vs each control. Bars are means + SEM of three to five independent experiments. *p<0.05 and ***p<0.001 vs each control, unpaired Student’s <i>t</i>-test. (C-H) MAP-2 immunostaining in control neuron-BV2 co-cultures (C, D) and co-cultures treated with LPS 100 ng/mL + IFN-γ 0.5 ng/mL for 24 h (E) or 48 h (G) and with LPS 100 ng/mL + IFN-γ 5 ng/mL for 24 h (F) or 48 h (H). Bar = 100 µm.</p

High extracellular K⁺ concentration did not potentiate reactive glia-induced neurotoxicity in neuron-primary microglia co-cultures.

<p>Neuronal viability (MAP2-ABTS-ELISA assay) in neuron-primary microglia co-cultures grown in 5 mM and 25 mM KCl and treated with 100 ng/mL LPS +30 ng/mL IFN-γ for 24 h. Results are presented as % of MAP2 immunostaining in control co-cultures grown in 5 mM KCl. Bars are means + SEM of four independent experiments. **p<0.01 vs each control; two-way ANOVA (repeated measures) and Bonferroni post-test.</p

The iNOS inhibitor 1400 W prevented the neurotoxicity induced by reactive microglia in neuron-primary microglia co-cultures.

<p>Neuron-primary microglia co-cultures were treated with 100 ng/mL LPS +30 ng/mL IFN-γ for 48 h in the presence or absence of 1400 W (10 µM) co-administered with LPS/IFN-γ. (A) NO production 48 h after LPS/IFN-γ treatment. (B) Evaluation of neuronal viability by MAP2-ABTS-ELISA assay. Results are presented as % of MAP2 immunostaining in control co-cultures. Bars are means + SEM of three independent experiments. **p<0.01 vs control; ##p<0.01 vs LPS/IFN-γ; one-way ANOVA (repeated measures) and Newman-Keuls post-test.</p

Glial activation and resulting neurotoxicity in neuron-primary microglia co-cultures.

<p>(A) Neuronal viability (MAP2-ABTS-ELISA assay) in neuronal cultures and neuron-primary microglia co-cultures 48 h after treatment with 100 ng/mL LPS and increasing concentrations of IFN-γ (15 and 30 ng/mL). (B) The addition of microglial cells at a microglia:neuron ratio of 1∶2 did not result in neurotoxicity after 48 h. Results are presented as % of MAP2 immunostaining vs each control. Bars are means + SEM of 3–4 independent experiments. *p<0.05 vs control; one-way ANOVA and Newman-Keuls post-test. MAP-2 immunocytochemistry in neuronal cultures (C-D) and neuron-primary microglia co-cultures (E-F) in control conditions (C and E) and 48 h after 100 ng/mL LPS +30 ng/mL IFN-γ (D and F).</p

Increased extracellular K⁺ concentration enhanced reactive glia-induced.

<p>neurotoxicity in neuron-BV2 co-cultures. (A) Neuronal viability (MAP2-ABTS-ELISA assay) in neuron-BV2 co-cultures grown in 5 mM and 25 mM KCl 24 h after 100 ng/mL LPS +0.5 ng/mL IFN-γ treatment. Results are presented as % of MAP2 immunostaining in control co-cultures grown in 5 mM KCl. Bars are means + SEM of four independent experiments. ***p<0.001 vs respective control; two-way ANOVA (repeated measures) and Bonferroni post-test. (B) MAP2 immunostaining in control (B) and 100 ng/mL LPS +0.5 ng/mL IFN-γ treated co-cultures grown in high K⁺. Bar = 100 µm. (D) Increasing BV2:neuron ratio results in neurotoxicity in control neuron-BV2 co-cultures. (E) Increasing BV2:neuron ratio does not result in increased neurotoxicity in 100 ng/mL LPS +0.5 ng/mL IFN-γ treated co-cultures.</p

IL-10 pre-treatment inhibited the pro-inflammatory response induced by LPS/IFN-γ in primary microglia cultures.

<p>Primary microglial cultures were treated with 100 ng/mL LPS +30 ng/mL IFN-γ for 24 h in the presence or absence of 50 ng/mL IL-10 administered 1 h prior to LPS/IFN-γ. TNF-α (A) and IL-6 (B) levels were determined in the culture medium 24 h after treatment. (C) COX-2 and (D) iNOS protein expression were measured by western blot 24 h after treatment, and data normalized by β-actin. (E) NO production was determined in the culture medium 24 h after treatment. Bars are means + SEM of three to four independent experiments. *p<0.05, **p<0.01 and ***p<0.001 vs control; ###p<0.001 vs LPS/IFN-γ; one-way ANOVA (repeated measures) and Newman-Keuls post-test. (F) shows a representative western blot.</p