Nurr1 expression is modified by inflammation in microglia

Abstract

Advances in neonatal care have allowed premature infants to survive at earlier gestational ages, but they are often afflicted with neurological delays or deficits. Maternal inflammation has been identified as a major risk factor for premature birth and once born, infants often require supplemental oxygen for survival. Nurr1 (NR4A2) is an orphan nuclear receptor with no known binding site and is essential for the growth of midbrain dopamine neurons. Others have reported that Nurr1 can act as an anti-inflammatory transcription factor in microglia and astrocytes and respond lipopolysaccharide (LPS). We have previously reported decreased numbers of oligodendrocytes and increased numbers of microglia in the mice exposed to both maternal inflammation and neonatal hyperoxia in the perinatal period. These studies tested the hypothesis that the combined exposures to inflammation and hyperoxia would increase Nurr1 expression in microglia in our mouse model and in an immortalized microglia cell line, BV2 cells. Our data indicate that Nurr1 protein expression is increased at postnatal day 0 and postnatal day 28 in whole-brain homogenates from mice exposed to LPS and hyperoxia. Alternatively, Nurr1 message is decreased at postnatal day 60 in isolated microglia, indicating that the increases in whole-brain homogenates may be due to other cell types. In BV2 cells, Nurr1 message in increased by exposure to hyperoxia, but this increase is attenuated in cells exposed to both LPS and hyperoxia. Although Nurr1 regulation is not straightforward, these data indicate that Nurr1 expression is increased in whole-brain homogenates in response to inflammation, but is decreased in isolated primary microglia and BV2 cells in response to similar inflammation. Our data support the hypothesis that Nurr1 expression may play a significant role in regulating inflammation in the brain and understanding the complex regulation of Nurr1 could lead to new therapeutic strategies.

Fig. 1

Nurr1 protein expression is increased in whole brain. Whole-brain homogenates were prepared from pups exposed to maternal LPS and/or neonatal hyperoxia at (a) PN day 0 and (b) PN day 28. Protein levels of Nurr1 were measured by Western blot and quantified by densitometry. Data were analyzed by two-way ANOVA, with Tukey’s post-hoc (*P<0.05, **P<0.01, n=5–8), and are presented as mean±SD. ANOVA, analysis of variance; LPS, lipopolysaccharide; PN, postnatal.

Fig. 2

Nurr1 and cytokine levels in microglia isolated from mice exposed to LPS and hyperoxia. Enriched microglia were isolated from mice exposed previously to maternal LPS and neonatal hyperoxia at PN60 as described in the Materials and methods section. RNA was isolated and cytokine expression was measured by RT-qPCR. Data were analyzed using a t-test (*P<0.05, n=4) and are presented as mean±SD. LPS, lipopolysaccharide; RA, room air; RT-qPCR, quantitative reverse transcription PCR.

Fig. 3

BV2 cell showed increased cytokine expression in response to LPS exposure. BV2 cells were exposed to LPS (50 ng/ml) or saline and exposed to room air or hyperoxia for 48 h (85% O₂). Cells were harvested and RNA extracted. Cytokine expression was measured by RT-qPCR. Data were analyzed using a t-test (**P<0.01, ***P<0.001, n=6) and are presented as mean±SD. ANOVA, analysis of variance; LPS, lipopolysaccharide; RA, room air; RT-qPCR, quantitative reverse transcription PCR.

Fig. 4

Combined effects of LPS and hyperoxia exposure decrease Nurr1 expression. BV2 cells were exposed to LPS (50 ng/ml) or saline and exposed to room air or hyperoxia for 48 h (85% O₂). Nonadherent cells and adherent cells were harvested independently and RNA were extracted. Nurr1 expression was measured by RT-qPCR. Data were analyzed by a two-way ANOVA with Tukey’s post-hoc, (*P<0.05, n=6) and are presented as mean±SD. ANOVA, analysis of variance; LPS, lipopolysaccharide; RT-qPCR, quantitative reverse transcription PCR.

Fig. 5

Combined LPS and hyperoxia exposure caused decreases in Nurr1 protein levels in BV2 cells. (a) BV2 cells were exposed to LPS (50 ng/ml) or saline and exposed to room air or hyperoxia for 48 h (85% O₂). Cells were harvested and cell lysates were analyzed by Western blot. Data were analyzed by a two-way ANOVA with Tukey’s post-hoc (*P<0.05, **P<0.01, n=6) and are presented as mean±SD. (b) Immunofluorescence was performed on BV2 cells exposed to LPS and hyperoxia. Images were taken at 400×, bar represents 50 µm. ANOVA, analysis of variance; LPS, lipopolysaccharide; RA, room air.