Corticosterone Production during Repeated Social Defeat Causes Monocyte Mobilization from the Bone Marrow, Glucocorticoid Resistance, and Neurovascular Adhesion Molecule Expression

Abstract

Repeated social defeat (RSD) stress promotes the release of bone marrow-derived monocytes into circulation that are recruited to the brain, where they augment neuroinflammation and cause prolonged anxiety-like behavior. Physiological stress activates the sympathetic nervous system and hypothalamic-pituitary-adrenal gland (HPA) axis, and both of these systems play a role in the physiological, immunological, and behavioral responses to stress. The purpose of this study was to delineate the role of HPA activation and corticosterone production in the immunological responses to stress in male C57BL/6 mice. Here, surgical (adrenalectomy) and pharmacological (metyrapone) interventions were used to abrogate corticosterone signaling during stress. We report that both adrenalectomy and metyrapone attenuated the stress-induced release of monocytes into circulation. Neither intervention altered the production of monocytes during stress, but both interventions enhanced retention of these cells in the bone marrow. Consistent with this observation, adrenalectomy and metyrapone also prevented the stress-induced reduction of a key retention factor, CXCL12, in the bone marrow. Corticosterone depletion with metyrapone also abrogated the stress-induced glucocorticoid resistance of myeloid cells. In the brain, these corticosterone-associated interventions attenuated stress-induced microglial remodeling, neurovascular expression of the adhesion molecule intercellular cell adhesion molecule-1, prevented monocyte accumulation and neuroinflammatory signaling. Overall, these results indicate that HPA activation and corticosterone production during repeated social defeat stress are critical for monocyte release into circulation, glucocorticoid resistance of myeloid cells, and enhanced neurovascular cell adhesion molecule expression.SIGNIFICANCE STATEMENT Recent studies of stress have identified the presence of monocytes that show an exaggerated inflammatory response to immune challenge and are resistant to the suppressive effects of glucocorticoids. Increased presence of these proinflammatory monocytes has been implicated in neuropsychiatric symptoms and the development of chronic cardiovascular, autoimmune, and metabolic disorders. In the current study, we show novel evidence that corticosterone produced during stress enhances the release of proinflammatory monocytes from the bone marrow into circulation, augments their recruitment to the brain and the induction of a neuroinflammatory profile. Overproduction of corticosterone during stress is also the direct cause of glucocorticoid resistance, a key phenotype in individuals exposed to chronic stress. Inhibiting excess corticosterone production attenuates these inflammatory responses to stress.

Keywords: HPA axis; corticosterone; inflammation; monocytes; repeated social defeat.

Figure 1.

Stress-induced release of inflammatory monocytes from the bone marrow into circulation was prevented by ADX. Male C57BL/6 mice were subjected to sham or ADX surgery and allowed to recover until exposure to RSD (Stress). Plasma for corticosterone was collected immediately after stress, and plasma for IL-6, bone marrow, and blood samples was collected 14 h later. A, Corticosterone levels (n = 9) and (B) IL-6 levels (n = 6) in the plasma, and (C) spleen weight (n = 9) were determined. D, Representative bivariate dot plots of monocytes (CD11b⁺ Ly6C^hi) and granulocytes (CD11b⁺ Ly6C^int) in the bone marrow. Percentage of bone marrow (E) monocytes and (F) granulocytes (n = 6). G, Representative bivariate dot plots of CD11b and Ly6C labeling of monocytes in circulation. H, Percentage of Ly6C^hi monocytes in the blood (n = 6). Error bars indicate mean ± SEM. Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other.

Figure 2.

Stress-associated reduction of CXCL12 in the bone marrow was attenuated by ADX. Male CXCL12-DsRed mice were exposed to control or RSD (Stress). A, Representative images of RFP expression in the femur immediately after stress (n = 3). Scale bar, 125 μm. Next, male C57BL/6 mice were exposed to control or RSD (Stress). B, CXCL12 mRNA expression in the bone marrow was determined (n = 3). In a separate experiment, male C57BL/6 mice were subjected to sham or ADX surgery and allowed to recover until exposure to RSD (Stress). C, CXCL12 mRNA expression in the bone marrow 14 h later (n = 6). Error bars indicate mean ± SEM. Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other.

Figure 3.

Stress-induced monocyte accumulation in the brain and the neurovascular induction of ICAM-1 were prevented by ADX. Male C57BL/6 mice were subjected to sham or ADX surgery and allowed to recover until exposure to RSD (Stress). Brain samples were collected 14 h later for flow cytometry and mRNA analyses. A, Representative bivariate dot plots. B, Percentage of CD45 and CD11b labeling of Percoll-enriched myeloid cells isolated from the brain (n = 6). mRNA expression of (C) TLR4, (D) IL-1β, and (E) ICAM-1 in a coronal brain section (n = 3–6). In a separate experiment, mice were treated as above. At 14 h after stress, brains were perfused, fixed, and labeled for ICAM-1 expression (n = 6). F, Representative images of ICAM-1 and Ly6C expression on blood vessels counterstained with DAPI in the dentate gyrus. Scale bar, 275 μm. Inset, Region used for analysis. Percentage area of ICAM-1 labeling in the (G) dentate gyrus and (H) PrL. Error bars indicate mean ± SEM. Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other. Inset, Region from which images were acquired.

Figure 4.

Stress-induced release of inflammatory monocytes from the bone marrow into circulation was attenuated by MTP. Male C57BL/6 mice were injected daily with either vehicle or MTP (100 mg/kg) 30 min before control or RSD (Stress). At 14 h after stress, brains were perfused, fixed, and labeled for ΔFosB. A, Representative images of ΔFosB expression in the prelimbic cortex (PrL). Scale bar, 125 μm. Inset, Region used for analysis. B, Average number of ΔFosB⁺ cells in the PrL (n = 3). C, Plasma corticosterone (n = 6). D, IL-6 levels (n = 3 or 4). E, Spleen weight (n = 6) was determined. Percentage of (F) CD11b⁺ Ly6C^hi monocytes and (G) CD11b⁺ Ly6C^int granulocytes in the bone marrow. H, Percentage and (I) representative bivariate dot plots of CD11b⁺ and Ly6C^hi labeling of monocytes in circulation. J, mRNA expression of CXCL12 in the bone marrow (n = 3 or 4). Error bars indicate mean ± SEM. Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other.

Figure 5.

MTP attenuated stress-induced glucocorticoid resistance of splenocytes. Male C57BL/6 mice were injected daily with either vehicle or MTP (100 mg/kg) 30 min before control or RSD (Stress). Splenocytes were collected 14 h later, and cultured ex vivo in presence of LPS (1 μg/ml) and increasing concentrations of corticosterone (0, 0.05, 0.1, 0.5, and 5 μm). A, Cell survival was determined 48 h after treatment, and results are expressed as percentage baseline of cell survival at 0 μm corticosterone (as indicated by the horizontal dashed line) (n = 6). B, Supernatant samples were collected from a duplicate preparation 18 h after the beginning of culture, and IL-6 levels were determined (n = 3). Error bars indicate mean ± SEM. Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other.

Figure 6.

MTP attenuated monocyte recruitment to the brain and prevented neuroinflammatory signaling. Male C57BL/6 mice were injected daily with either vehicle or MTP (100 mg/kg) 30 min before control or RSD (Stress). At 14 h after stress, brains were perfused, fixed, and labeled for Iba-1. A, Representative images of Iba-1 expression in the dentate gyrus. Scale bar, 275 μm. Inset, Region used for analysis. Percentage area of Iba-1 labeling in the (B) dentate gyrus and (C) PrL (n = 3 or 4). In a separate experiment, mice were treated as above. D, Representative bivariate dot plots of CD45 and CD11b labeling of Percoll-enriched myeloid cells isolated from the brain. E, The percentage of CD45^hi monocytes/macrophages in the enriched CD11b⁺ from the brain (n = 3). mRNA expression of (F) TLR4, (G) IL-1β, and (H) ICAM-1 in a coronal brain section (n = 3–6). Error bars indicate mean ± SEM. Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other.

Figure 7.

MTP prevented stress-induced induction of endothelial ICAM-1 in the brain. Male C57BL/6 mice were injected daily with either vehicle or MTP (100 mg/kg) 30 min before control or RSD (Stress). At 14 h after stress, brains were perfused, fixed, and labeled for ICAM-1 expression. A, Representative images of ICAM-1 and Ly6C expression on blood vessels counterstained with DAPI in the dentate gyrus. Scale bar, 275 μm. Inset, Region used for analysis. Percentage area of ICAM-1 labeling in the (B) dentate gyrus and (C) prelimbic cortex (PrL) (n = 3 or 4). Error bars indicate mean ± SEM. Means with different letters (a, b, or c) are significantly different (p < 0.05) from each other.