Yufen Wang

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

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.

Neural and behavioral responses to low-grade inflammation.

Neural and behavioral responses after peripheral immune challenge have been observed in numerous studies. The majority of these studies have utilized relatively high doses of lipopolysaccharide (LPS) as the immune stimulant. Little attention has been given to the effects of LPS dose ranges that simulate low grade-inflammation. The current studies were designed to characterize neural and behavioral responses following low-dose LPS stimulation. Results show burrowing and open field activity was significantly impaired following a single i.p. injection of 10, but not 1, μg/kg of LPS. In addition, following repeated 1 μg/kg LPS administration for 10 days, animals showed the progressive development of motor deficits over time. To correlate behavior with CNS activity, cFos activation was determined in the paraventricular nucleus, nucleus of the solitary tract, central amygdaloid nucleus, and ventrolateral medulla. Data revealed there was a dose-dependent activation in all brain areas examined, but only the PVN showed significant activation by low-dose LPS. Additionally, animals that received 1 μg/kg of LPS for 8 days had PVN cFos activation similar to animals that received a single 10 μg/kg LPS injection. These data demonstrate neural and behavior responses can be induced by low-grade inflammation and chronic exposure to sub-threshold levels of LPS can precipitate significantly heightened neural and behavioral responses.

Neuronal and Nonneuronal COX-2 Expression Confers Neurotoxic and Neuroprotective Phenotypes in Response to Excitotoxin Challenge

Treating acute brain injuries with COX‐2 inhibitors can produce both neuroprotective and neurotoxic effects. This study investigated the role of COX‐2 in modulating acute brain injury induced by excitotoxic neural damage. Intrastriatal injection of excitotoxin (RS)‐(tetrazole‐5yl) glycine elicited COX‐2 expression in two distinct groups of cells. cortical neurons surrounding the lesion and vascular cells in the lesion core. The vascular COX‐2 was expressed in two cell types, endothelial cells and monocytes. Selective deletion of COX‐2 in vascular cells in Tie2Cre Cox‐2flox/flox mice did not affect the induction of COX‐2 in neurons after the excitotoxin injection but resulted in increased lesion volume, indicating a neuroprotective role for the COX‐2 expressed in the vascular cells. Selective deletion of monocyte COX‐2 in LysMCre Cox‐2flox/flox mice did not reduce COX‐2‐dependent neuroprotection, suggesting that endothelial COX‐2 is sufficient to confer neuroprotection. Pharmacological inhibition of COX‐2 activity in Tie2Cre Cox‐2flox/flox mice reduced lesion volume, indicating a neurotoxic role for the COX‐2 expressed in neurons. Furthermore, COX‐2‐dependent neurotoxicity was mediated, at least in part, via the activation of the EP1 receptor. These results show that Cox‐2 expression induced in different cell types can confer opposite effects.

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

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.

Controlled progressive innate immune stimulation regimen prevents the induction of sickness behavior in the open field test.

Peripheral immune activation by bacterial mimics or live replicating pathogens is well known to induce central nervous system activation. Sickness behavior alterations are often associated with inflammation-induced increases in peripheral proinflammatory cytokines (eg, interleukin [IL]-1β and IL-6). However, most researchers have used acute high dose endotoxin/bacterial challenges to observe these outcomes. Using this methodology may pose inherent risks in the translational interpretation of the experimental data in these studies. Studies using Escherichia coli have yet to establish the full kinetics of repeated E. coli peripheral injections. Therefore, we sought to examine the effects of repeated low dose E. coli on sickness behavior and local peripheral inflammation in the open field test. Results from the current experiments showed a behavioral dose response, where increased amounts of E. coli resulted in correspondingly increased sickness behavior. Furthermore, animals that received a subthreshold dose (ie, one that did not cause sickness behavior) of E. coli 24 hours prior were able to withstand a larger dose of E. coli on the second day (a dose that would normally cause sickness behavior in mice without prior exposure) without inducing sickness behavior. In addition, animals that received escalating subthreshold doses of E. coli on days 1 and 2 behaviorally tolerated a dose of E. coli 25 times higher than what would normally cause sickness behavior if given acutely. Lastly, increased levels of E. coli caused increased IL-6 and IL-1β protein expression in the peritoneal cavity, and this increase was blocked by administering a subthreshold dose of E. coli 24 hours prior. These data show that progressive challenges with subthreshold levels of E. coli may obviate the induction of sickness behavior and proinflammatory cytokine expression.

Prostacyclin mediates endothelial cOX-2- dependent neuroprotective effects during excitotoxic brain injury

In a previous study, we found that intracerebral administration of excitotoxin (RS)-(tetrazole-5yl) glycine caused increased neural damage in the brain in an endothelial COX-2 deleted mouse line (Tie2Cre COX-2flox/flox). In this study, we investigated whether prostacyclin might mediate this endothelial COX-2-dependent neuroprotection. Administration of excitotoxin into the striatum induced the production of prostacyclin (PGI2) in wild type, but not in endothelial COX-2 deleted mice. Inhibition of PGI2 synthase exacerbated brain lesions induced by the excitotoxin in wild type, but not in endothelial COX-2 deleted mice. Administration of a PGI2 agonist reduced neural damage in both wild type and endothelial COX-2 deleted mice. Increased PGI2 synthase expression was found in infiltrating neutrophils. In an ex vivo assay, PGI2 reduced the excitotoxin-induced calcium influx into neurons, suggesting a cellular mechanism for PGI2 mediated neuroprotection. These results reveal that PGI2 mediates endothelial COX-2 dependent neuroprotection.

64. Roles of glucocorticoid receptor and microRNA in repeated social disruption stress-induced glucocorticoid resistance in splenic macrophages

Chronic social stress has attracted interest due to its deleterious impact on health psychologically and physiologically. It has been shown using rodent models of chronic social stress, e.g., repeated social disruption stress (RSD), innate immune cells develop insensitivity to glucocorticoid (GC) stimuli and contribute to enhance inflammatory responses. Previous studies have found that one of the immune cells that is GC insensitive in response to RSD is the splenic macrophage, however, the underlying molecular mechanisms that contribute to this process are not fully understood. Current data showed RSD modified GC receptor expression and increased the expression ratio of FK506-binding protein 51 (FKBP51) to FKBP52, favoring to blockade of nuclear translocation of GC–GC receptor complexes in splenic macrophages. Using a NanoString nCounter miRNA Expression assay and the internet-based DIANA-micro T-CDS database (ver 5.0), the expression of 6 microRNAs was increased by RSD. The two highest expressed miRNAs (miRNA-29b and 340) were overexpressed in L929 cells and were found to completely block LPS-induced overexpression of GC receptors. These data suggest that miRNAs play a critical role in RSD-induced GC resistance in splenic macrophages, especially via an epigenetic mechanism of GC receptor expression at least. This was supported by grants funded through NIH/NIMH: R01MH093473 and R01MH097243.

71. Infiltrating PGI2 producing leukocytes mediate neuroprotecitive effect of cyclooxygenase-2 (COX-2)

We showed previously that COX-2 is induced in the brain by excitotoxin in two distinct cell populations, neurons and non-neuronal cells. When cox-2 gene is selectively deleted in non-neuronal cells in a conditional knockout mouse line (KO), brain injury volumes induced by excitotoxin were nearly twice as large as those in WT controls, suggesting that the COX-2 expression in non-neuronal cells plays a neuroprotective role. In this study, we investigated the cellular and molecular mechanisms underlying COX-2-dependent neuroprotection. Correlated with the smaller lesion in WT mice, PGI2-producing leukocytes were found to infiltrate into the brain parenchyma in and near the site of lesion in WT, but not the KO, mice. At the site of injury, the level of PGI2 increased significantly after excitotoxin injection in WT, but not the KO, mice. In addition, inhibition of the PGI2 synthesis in WT animals induced an increase in the lesion size, whereas injection of PGI2 agonist in the KO mice reduced the lesion size. Further, after receiving adoptive transfer of blood leukocytes from WT mice, PGI2-expressing cells were found at the site of excitotoxic lesion in the KO animals with a concomittant reduction of lesion volume. These data suggest that PGI2 produced by the infiltrating leukocytes mediates the neuroprotective effect of the non-neuronal expression of COX-2.

The induction of COX-2 in different cell types plays different roles in excitotoxic neuronal cell death

wound site, increasing the risk of opportunistic infection. Toll-like receptor 4 (TLR4) signaling pathway, which is crucial for bacterial clearance, is regulated by small non-coding RNA called microRNAs (miRs). We hypothesis that stress induces differential expression of miRs that regulate TLR4 pathway contributing to impaired inflammation and bacterial clearance during wound healing. SKH-1 mice were either food-and-water (FWD) deprived to serve as controls or stressed by restraint (RST) 12 h/day for 3 days prior and 5 days post-wounding. Two punch biopsy wounds of 3.5 mm were created on their backs. Wounds were harvested for each condition 0, 1 and 5 days post-wounding and processed for qRT-PCR and miR profiling (n = 5) to determine the expression of members of TLR4 pathway and TLR4-related miRs, respectively. We observed that stress down-regulates the expression of molecules involved in TLR4 signaling cascade at day 1 post-wounding compared to day 5 in controls. TLR4-related miRs levels change during the course of wound healing, and they are differentially regulated under stress. MiR-21 and its target, PDCD4, show inverse expression profiles in controls, whereas miR-146b and IRAK1 levels follow opposite trends in stress-impaired wound healing. Our study suggests that stress affects miRs regulating members of TLR4 signaling cascade, dampening their expression, which could compromise the inflammatory response and bacterial clearance during stress-impaired wound healing.