Mark L. Hanke

Minocycline attenuates lipopolysaccharide (LPS)-induced neuroinflammation, sickness behavior, and anhedonia

BackgroundActivation of the peripheral innate immune system stimulates the secretion of CNS cytokines that modulate the behavioral symptoms of sickness. Excessive production of cytokines by microglia, however, may cause long-lasting behavioral and cognitive complications. The purpose of this study was to determine if minocycline, an anti-inflammatory agent and purported microglial inhibitor, attenuates lipopolysaccharide (LPS)-induced neuroinflammation, sickness behavior, and anhedonia.MethodsIn the first set of experiments the effect of minocycline pretreatment on LPS-induced microglia activation was assessed in BV-2 microglia cell cultures. In the second study, adult (3–6 m) BALB/c mice received an intraperitoneal (i.p.) injection of vehicle or minocycline (50 mg/kg) for three consecutive days. On the third day, mice were also injected (i.p.) with saline or Escherichia coli LPS (0.33 mg/kg) and behavior (i.e., sickness and anhedonia) and markers of neuroinflammation (i.e., microglia activation and inflammatory cytokines) were determined. In the final study, adult and aged BALB/c mice were treated with the same minocycline and LPS injection regimen and markers of neuroinflammation were determined. All data were analyzed using Statistical Analysis Systems General Linear Model procedures and were subjected to one-, two-, or three-way ANOVA to determine significant main effects and interactions.ResultsMinocycline blocked LPS-stimulated inflammatory cytokine secretion in the BV-2 microglia-derived cell line and reduced LPS-induced Toll-like-receptor-2 (TLR2) surface expression on brain microglia. Moreover, minocycline facilitated the recovery from sickness behavior (i.e., anorexia, weight loss, and social withdrawal) and prevented anhedonia in adult mice challenged with LPS. Furthermore, the minocycline associated recovery from LPS-induced sickness behavior was paralleled by reduced mRNA levels of Interleukin (IL)-1β, IL-6, and indoleamine 2, 3 dioxygenase (IDO) in the cortex and hippocampus. Finally, in aged mice, where exaggerated neuroinflammation was elicited by LPS, minocycline pretreatment was still effective in markedly reducing mRNA levels of IL-1β, TLR2 and IDO in the hippocampus.ConclusionThese data indicate that minocycline mitigates neuroinflammation in the adult and aged brain and modulates the cytokine-associated changes in motivation and behavior.

β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat.

Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b+/CD45high/Ly6Chigh macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors.

Repeated social defeat activates dendritic cells and enhances Toll-like receptor dependent cytokine secretion

Stress hormones significantly impact dendritic cell (DC) activation and function, typically in a suppressive fashion. However, a social stressor termed social disruption (SDR) has been shown to induce an increase in inflammatory responses and a state of glucocorticoid resistance in splenic CD11b+ monocytes. These experiments were designed to determine the effects of SDR on DC activation, Toll-like receptor-induced cytokine secretion, and glucocorticoid sensitivity. Compared to cells obtained from control animals, splenic DCs from SDR mice displayed increased levels of MHC I, CD80, and CD44, indicative of an activated phenotype. In addition, DCs from SDR mice produced comparatively higher TNF-alpha, IL-6, and IL-10 in response to in vitro stimulation with LPS and CpG DNA. Increased amounts of TNF-alpha and IL-6 were also evident in SDR DC cultures stimulated with poly(I:C). Furthermore, as shown previously in CD11b+ monocytes, the CD11c+ DCs obtained from SDR mice were glucocorticoid resistant. Taken together, the data suggest that social stress, in the absence of any immune challenge, activates DCs, increases DC cytokine secretion in response to Toll-specific stimuli and renders DCs glucocorticoid resistant.

Beta adrenergic blockade decreases the immunomodulatory effects of social disruption stress

During physiological or psychological stress, catecholamines produced by the sympathetic nervous system (SNS) regulate the immune system. Previous studies report that the activation of β-adrenergic receptors (βARs) mediates the actions of catecholamines and increases pro-inflammatory cytokine production in a number of different cell types. The impact of the SNS on the immune modulation of social defeat has not been examined. The following studies were designed to determine whether SNS activation during social disruption stress (SDR) influences anxiety-like behavior as well as the activation, priming, and glucocorticoid resistance of splenocytes after social stress. CD-1 mice were exposed to one, three, or six cycles of SDR and HPLC analysis of the plasma and spleen revealed an increase in catecholamines. After six cycles of SDR the open field test was used to measure behaviors characteristic of anxiety and indicated that the social defeat induced increase in anxiety-like behavior was blocked by pre-treatment with the β-adrenergic antagonist propranolol. Pre-treatment with the β-adrenergic antagonist propranolol did not significantly alter corticosterone levels indicating no difference in activation of the hypothalamic-pituitary-adrenal axis. In addition to anxiety-like behavior the SDR induced splenomegaly and increase in plasma IL-6, TNFα, and MCP-1 were each reversed by pre-treatment with propranolol. Furthermore, flow cytometric analysis of cells from propranolol pretreated mice reduced the SDR-induced increase in the percentage of CD11b(+) splenic macrophages and significantly decreased the expression of TLR2, TLR4, and CD86 on the surface of these cells. In addition, supernatants from 18h LPS-stimulated ex vivo cultures of splenocytes from propranolol-treated SDR mice contained less IL-6. Likewise propranolol pre-treatment abrogated the glucocorticoid insensitivity of CD11b(+) cells ex vivo when compared to splenocytes from SDR vehicle-treated mice. Together, this study demonstrates that the immune activation and priming effects of SDR result, in part, as a consequence of SNS activation.

Social disruption induces lung inflammation

Social disruption (SDR) is a well-characterized mouse stressor that causes changes in immune cell reactivity in response to inflammatory stimuli. In this study, we found that SDR in the absence of an immune challenge induced pulmonary inflammation and increased pulmonary myeloperoxidase activity. The percentage of neutrophils within the lungs increased 2-fold after social disruption. Monocyte accumulation in the lungs was also significantly increased. In addition, SDR increased the percentage of neutrophils that expressed CD11b, indicating that more neutrophils were in an activated state. In the lungs, we observed an increased level of the inflammatory cytokine, IL-1beta, as well as higher levels of KC/CXCL1, MIP-2/CXCL2, and MCP-1/CCL2, which are chemokines responsible for neutrophil and monocyte recruitment. Furthermore, social disruption led to increased lung expression of the adhesion molecules P-selectin, E-selectin, and ICAM-1, which localize and recruit immune cells. These data support previous findings of an inflammatory environment induced by SDR. We demonstrate that this effect also occurs in the pulmonary milieu and in the absence of an inflammatory stimulus.

Immunogenic dendritic cells primed by social defeat enhance adaptive immunity to influenza A virus

Dendritic cells (DCs) sample their surrounding microenvironment and consequently send immunogenic or regulatory signals to T cells during DC/T cell interactions, shaping the primary adaptive immune response to infection. The microenvironment resulting from repeated social defeat increases DC co-stimulatory molecule expression and primes DCs for enhanced cytokine responses in vitro. In this study, we show that social disruption stress (SDR) results in the generation of immunogenic DCs, capable of conferring enhanced adaptive immunity to influenza A/PR/8/34 infection. Mice infected with influenza A/PR/8/34 virus 24 h after the adoptive transfer of DCs from SDR mice had significantly increased numbers of D(b)NP(366-74)CD8(+) T cells, increased IFN-γ and IFN-α mRNA, and decreased influenza M1 mRNA expression in the lung during the peak primary response (9 days post-infection), compared to mice that received DCs from naïve mice. These data demonstrate that repeated social defeat is a significant environmental influence on immunogenic DC activation and function.

Stress and the anti-influenza immune response: Repeated social defeat augments clonal expansion of CD8 + T cells during primary influenza A viral infection

Social disruption stress (SDR) prior to primary influenza A virus (IAV) infection augments memory to IAV re-challenge in a T cell-specific manner. However, the effect of SDR on the primary anti-viral immune response has not been elucidated. In this study, SDR-infected (INF) mice terminated viral gene expression earlier and mounted an enhanced pulmonary IAV-specific CD8(+)T cell response versus controls. Additionally, SDR-INF mice had a more pro-inflammatory lung profile prior to and during infection and an attenuated corticosterone response. These data demonstrate neuroendocrine modification of the lung microenvironment and increased antigen-specific T cell activation, clonal expansion and viral control in stress-exposed mice.

Repeated social defeat increases catecholamine production in the spleen, liver, mediastinal lymph nodes, and brain of CD-1 mice

TSD challenge. Habitual sleep duration did not predict increase in fatigue (r = .34, n.s.). Furthermore, subjects with short habitual sleep duration tended to have a higher monocyte increase in response to a TSD challenge then those with longer sleep duration (r = .46, p < .07). Conclusion: In pain-free, healthy subjects, prolonged TSD led to a 9%-increase of subjective pain. Increase in pain was more pronounced in subjects with shorter habitual sleep durations, suggesting that habitual sleep duration may contribute to interindividual variability in the pain response to TSD.