Molecular mechanisms of repeated social defeat-induced glucocorticoid resistance: Role of microRNA

Abstract

Glucocorticoid (GC) resistance is a severe problem associated with various inflammatory diseases. Previous studies have shown that repeated social stress induces GC resistance in innate immune cells, but the underlying molecular mechanisms have not been fully elucidated. Therefore, the purpose of this study was to examine potential underlying molecular mechanism(s) of repeated social defeat (RSD) stress on GC resistance in splenic macrophages. It was hypothesized that mRNA expression of receptors for GC and nuclear translocating-associated regulators in splenic macrophages would be affected by RSD, and that these changes would be associated with epigenetic modification. The data showed that the mRNA expression of GC and mineralocorticoid receptors were significantly decreased in splenic macrophages by RSD. RSD also induced a significantly decreased mRNA expression in FK506-binding protein 52 (FKBP52), consequently resulting in a significantly increased ratio of FKBP51 to FKBP52. Moreover, DNA methyltransferases 3a and 3b showed a significant decrease in their mRNA expression in the RSD group as did mRNA expression of histone deacetyltransferase 2. The RSD group also showed a significantly reduced quantity of methylated DNA in splenic macrophages. Based on microRNA (miRNA) profiling data, it was determined that RSD induced significantly increased expression of 9 different miRNAs that were predicted to interact with mRNAs of the GC receptor (6 miRNAs), mineralocorticoid receptor (3 miRNAs) and FKBP52 (2 miRNAs). Spearman correlation analysis revealed significantly strong correlations between the expression of 2 miRNAs and their target mRNA expression for GC receptors. Among these miRNAs, we verified direct effects of miRNA-29b and -340 overexpression on mRNA expression of GC receptors in L929 cells. The overexpression of miRNA-29b or -340 in L929 cells significantly reduced LPS-induced overexpression of GC receptors. In conclusion, this study provides evidence that epigenetic regulation, such as DNA methylation and miRNA expression, may play a role in the RSD-induced GC resistance that we have observed in splenic macrophages.

Keywords: Chronic social stress; Epigenetics; Glucocorticoid insensitivity; Innate immunity; Macrophages; miRNA.

Figure 1

Effects of the 6-cycles of RSD on expression of glucocorticoid-associated proteins in splenic macrophages (CD11b⁺ cells) in the control (clear bar) and RSD (filled bar) groups. (A) mRNA expression of glucocorticoid (nr3c1) and mineralocorticoid (nr3c2) receptors (n = 8/group). Equal Variance F test for the dependent variable of mineralocorticoid receptors failed (p <0.05). The median values for the control and RSD groups were 1.057 and 0.160 respectively, and the p value calculated by two-tailed unequal variance t test was 0.0015 (mean ± SEM). (B) Results of FKBP51 (fkbp5) and FKBP52 (fkbp4) expression (n = 6/group). Equal Variance F test for the dependent variable of mineralocorticoid receptors failed (p <0.05). The median values for the control and RSD groups were 0.988 and 0.751 respectively, and the p value calculated by two-tailed unequal variance t test was 0.139 (mean ± SEM). (C) Ratio of FKBP51 (fkbp5) and FKBP52 (fkbp4) mRNA expression (mean ± SEM). (D) Gating examples of CD11b and GC receptor on splenocytes in control and RSD groups. Splenocytes were first gated based on plotting FSC vs SCC (top), and flow cytometric analyses of CD11b and GC receptor expression were performed (bottom). (E) Protein expression of GC receptors (n = 6/group) on splenic macrophages. Protein expression of GC receptors in CD11b⁺ splenocytes was determined by using MFI of GC receptors, and the p value was calculated by two-tailed equal variance t test (mean ± SEM). #: p = 0.003; *: p ≤ 0.001; **: p ≤ 0.0001

Figure 2

Effects of the 6-cycles of RSD on mRNA expression of chromatin modification-associated proteins and status of DNA methylation in splenic macrophages (CD11b⁺ cells). (A) mRNA expression of DNA methyltransferases DNMT3a, DNMT3b, and DNMT1 in the control (clear bar) and RSD (filled bar) groups (mean ± SEM, n = 8/group) (B) mRNA expression of histone deacetylase 2 (mean ± SEM) (C) Standard curve plot for OD values observed at 450 nm vs. concentrations of control methylated DNA (5-mC) The coefficient of determination (R²) for the linear regression is 0.9947. (D) The absolute amount of methylated DNA in splenic macrophages (CD11b⁺ cells) in control and RSD groups (mean ± SEM) *: p< 0.001; **: p< 0.0001

Figure 3

Effects of the 6-cycles of RSD on miRNA expression in splenic macrophages (CD11b⁺ cells) and effects of miRNA overexpression in glucocorticoid receptor expression in L929 cells. (A) The heatmap for selected miRNAs that were recognized from the DIANA-microT-CDS database (Ver. 5.0, http://www.microrna.gr/microT-CDS) and also showed significant differences in their expression between control and RSD groups (n = 5/group, p< 0.05 by two-tailed t test). A miRNA with less than 50 counts from both control and RSD was excluded. The heatmap (cell plot) was generated by JMP® statistical discovery software (SAS Institute Inc., Ver. 10.0.2, 64 bit Edition) with a Green-to-Black-to-Red color theme. The miTG score (prediction score) for each miRNA was given in parentheses. (B), (C) Spearman correlation coefficients of miRNA expression with their target mRNAs. The expression of miRNA-29b (B) and miRNA-340 (C) were all significantly correlated with the expression of their target mRNAs for glucocorticoid receptor (GR, nr3c1). Linear regression equations for each miRNA expression with glucocorticoid receptor (y) were included on the plot with Spearman correlation coefficients (ρ) and p values (n = 14). (D) Verification of two selected miRNA expression in splenic macrophages in the control (clear bar) and RSD (filled bar) groups (mean ± SEM, n = 8/group). miRNAs were chosen if it was predicted to interact with nr3c1 mRNA with more than 100 counts in both groups. The expression level of each miRNA was normalized to that of the SYBR Green detection. *: p= 0.00271; **: p< 0.0001 (E) Effects of miRNA-29b and 340 overexpression on glucocorticoid receptor (nr3c1) expression in L929 cells (mean ± SEM, n = 4/group). One way ANOVA test resulted in a significant group difference (p < 0.001), and post-hoc test (Student-Newman-Keuls Method) was performed (*: p< 0.01 vs. control, vehicle control, LPS-control, and LPS-vehicle control groups).

Figure 4

Diagram of the hypothesized underlying mechanisms for the RSD-induced glucocorticoid resistance in splenic macrophages (CD11b⁺ cells). For the glucocorticoid resistance induced by chronic social stress, chromatin modification includes DNA and histone methylation/demethylation and histone acetylation/deacetylation based on the present study.