Repeated social defeat causes increased anxiety-like behavior and alters splenocyte function in C57BL/6 and CD-1 mice

The experimental model, social disruption (SDR), is a model of social stress in which mice are repeatedly attacked and defeated in their home cage by an aggressive conspecific. In terms of the impact of this stressor on the immune response, SDR has been reported to cause hyperinflammation and glucocorticoid insensitivity. To this point however, the behavioral consequences of SDR have not been thoroughly characterized. Because social defeat has been reported to cause anxiety- and depressive-like behaviors, the current study was designed to assess whether SDR also causes anxiety- and depressive-like behaviors. Using the light/dark preference test and the open field test as tools to measure behaviors characteristic of anxiety, the data showed that C57BL/6 and CD-1 male mice subjected to SDR displayed increased anxiety-like behavior. The increase in anxiety-like behaviors persisted for at least 1 week after the cessation of the stressor. In contrast, depressive-like behaviors were not elicited by SDR as assessed by the forced swim test or the tail suspension test. These data indicate that social disruption stress causes an increase in anxiety-like behaviors, but not depressive-like behaviors.     

β-Adrenergic receptor mediated increases in activation and function of natural killer cells following repeated social disruption

Natural killer (NK) cells are specialized innate lymphocytes important in the early defense against tumor and virus bearing cells. Many factors influence the immune system’s effectiveness against pathogens, including stress. Social disruption (SDR) “primes” macrophages/monocytes and dendritic cells thereby enhancing their anti-microbial function. What remains unclear is whether similar responses are evident in NK cells. Current studies investigated the cellular distribution and activation/inhibitory phenotypes of NK cells in the spleen, lung, and blood of C57BL/6 male mice following SDR. Furthermore, cytolytic activity and anti-viral cytokine production of splenic NK cells were determined. Lastly, β-adrenergic receptor (β-AR) signaling was investigated to determine possible mechanisms behind the SDR-induced NK cell alterations. Results indicated NK cells from SDR mice have increased expression of CD16 and CD69 and reduced NKG2a and Ly49a expression on splenic CD3?/DX5+ NK cells indicative of an activated phenotype, both immediately and 14 h post-SDR. Administration of propranolol (10 mg/kg; non-selective β-adrenergic receptor antagonist) was shown to block these “priming” effects at the 14 h time-point. In the lung, SDR had similar effects on activation and inhibitory receptors 14 h post-SDR, however no alterations were evident in the blood besides increased NK cells directly after SDR. Additionally, splenic NK cells from SDR mice had increased CD107a surface expression, cytolytic activity, and IFN-γ production was increased upon costimulation with IgG and IL-2 ex vivo. Collectively, these data suggest that social stress “primes” NK cells in the spleen and lung to be more proficient in their cytolytic and anti-viral/tumor effecter functions through β-adrenergic receptor dependent signaling.     

Peripheral innate immune challenge exaggerated microglia activation, increased the number of inflammatory CNS macrophages, and prolonged social withdrawal in socially defeated mice

Repeated social defeat (RSD) activates neuroendocrine pathways that have a significant influence on immunity and behavior. Previous studies from our lab indicate that RSD enhances the inflammatory capacity of CD11b? cells in the brain and promotes anxiety-like behavior in an interleukin (IL)-1 and β-adrenergic receptor-dependent manner. The purpose of this study was to determine the degree to which mice subjected to RSD were more responsive to a secondary immune challenge. Therefore, RSD or control (HCC) mice were injected with saline or lipopolysaccharide (LPS) and activation of brain CD11b? cells and behavioral responses were determined. Peripheral LPS (0.5 mg/kg) injection caused an extended sickness response with exaggerated weight loss and prolonged social withdrawal in socially defeated mice. LPS injection also amplified mRNA expression of IL-1β, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), and CD14 in enriched CD11b? cells isolated from socially defeated mice. In addition, IL-1β mRNA levels in enriched CD11b? cells remained elevated in socially defeated mice 24 h and 72 h after LPS. Moreover, microglia and CNS macrophages isolated from socially defeated mice had the highest CD14 expression after LPS injection. Both social defeat and LPS injection increased the percentage of CD11b?/CD45(high) macrophages in the brain and the number of inflammatory macrophages (CD11b?/CD45(high)/CCR2?) was highest in RSD-LPS mice. Anxiety-like behavior was increased by social defeat, but was not exacerbated by the LPS challenge. Nonetheless, reduced locomotor activity and increased social withdrawal were still present in socially defeated mice 72 h after LPS. Last, LPS-induced microglia activation was most evident in the hippocampus of socially defeated mice. Taken together, these findings demonstrate that repeated social defeat enhanced the neuroinflammatory response and caused prolonged sickness following innate immune challenge.     

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.     

Social stress and the regulation of tumor necrosis factor-alpha secretion

Social disruption (SDR), a murine model of social stress, altered the phenotype and function of spleen immune cells. Previous reports indicated that following SDR spleens contained higher numbers of CD11b+ monocytes, and these cells were less sensitive to the inhibitory effects of glucocorticoids on cell viability. Additionally, lipopolysaccharide (LPS)-stimulated splenocytes from SDR mice secreted higher levels of the proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 compared to splenocytes from controls. The present study sought to further examine the effects of SDR on TNFalpha secretion from splenocytes. We report that SDR increased TNFalpha secretion from an enriched fraction of CD11b+ monocytes stimulated with LPS. Additionally, SDR altered the kinetics of TNFalpha release from LPS-stimulated splenocytes and induced minor changes in the suppressive effects of corticosterone and norepinephrine on LPS-induced TNFalpha secretion. These results are in agreement with the notion that complex interactions mediate the response to social stress.     

Repeated social defeat in female mice induces anxiety-like behavior associated with enhanced myelopoiesis and increased monocyte accumulation in the brain

Anxiety and mood disorders affect both men and women. The majority of experimental models of stress, however, are completed using only male animals. For repeated social defeat (RSD), a rodent model, this is due to the inherent difficulty in eliciting male aggression toward female mice. To address this limitation, a recent study showed that a DREADD-based activation of the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) was effective in inducing aggressive behavior in male mice towards females in a social defeat paradigm. Therefore, the goal of this study was to determine if this modified version of RSD in females elicited behavioral, physiological, and immune responses similar to those reported in males. Here, we show that female mice subjected to RSD with the male DREADD aggressor developed anxiety-like behavior and social avoidance. These behavioral alterations coincided with enhanced neuronal and microglial activation in threat-appraisal regions of the brain. Moreover, stressed female mice had an enhanced peripheral immune response characterized by increased myelopoiesis, release of myeloid cells into circulation, and monocyte accumulation in the spleen and brain. These results are consistent with previously reported findings that male mice exposed to RSD exhibited increased fear and threat appraisal responses, enhanced myelopoiesis, myeloid cell release and trafficking, and anxiety-like behavior. These findings validate that RSD is a relevant model to study stress responses in female mice.     

Psychological stress increases expression of IL-10 and its homolog IL-19 via β-adrenoceptor activation: Reversal by the anxiolytic chlordiazepoxide

Recent studies from our laboratory indicate that psychological stress is a potent inducer of the anti-inflammatory cytokine interleukin (IL)-10, raising the possibility that the IL-10 family of cytokines may be key mediators of stress-induced immunosuppression. In this study we examined the impact of psychological stress (restraint stress) on expression of IL-10, and the novel IL-10 family members IL-19, IL-20 and IL-24 in mouse spleen following an in vivo challenge with lipopolysaccharide (LPS). We found that stressor exposure significantly augmented LPS-induced IL-10 expression. Similarly, IL-19 expression was induced by LPS, and this was significantly enhanced by restraint stress. In contrast, expression of IL-24 was not significantly altered by LPS or stress, and expression of IL-20 was largely not detectable in vivo in either saline or LPS-treated animals. Consistent with a role for sympathetic nervous system (SNS) activation in stress-induced immune regulation, the sympathetic neurotransmitter noradrenaline increased LPS-induced IL-10 and IL-19 expression in splenocytes and dendritic cells, and the ability of noradrenaline to induce expression of these cytokines was blocked by pre-treatment with the β-adrenoceptor antagonist propranolol. Similarly, pre-treatment of mice with the peripherally acting β-adrenoceptor antagonist nadolol completely blocked the stress-induced increase in IL-10 and IL-19 mRNA expression. Finally, pre-treatment with the benzodiazepine anxiolytic chlordiazepoxide prevented the stress-induced increase in IL-10 and IL-19 expression. Taken together, these data demonstrate that psychological stress induces expression of the IL-10 and its homolog IL-19 via activation of β-adrenoceptors, and the ability of stress to induce these cytokines is prevented by treatment with the anxiolytic chlordiazepoxide. The findings suggest that stress enhances the production of immunosuppressive cytokines, which may impact on stress-related disease processes.     

Social stress alters splenocyte phenotype and function

Social stress of group-housed male mice induced a state of functional glucocorticoid (GC) resistance in splenocytes. The following studies examined the effects of paired-fighting (PF) stress on immune cell distribution and function in spleens of male mice. Following six daily PF stress sessions, splenic monocytes and neutrophils increased and lymphocytes decreased. PF also altered the distribution of CD62L and CD11b positive monocytes. Additionally, PF augmented proliferation and lowered the sensitivity of LPS-stimulated splenocytes to the antiproliferative effects of corticosterone, suggesting that PF induced a state of GC resistance in splenocytes. Together, these findings indicate that social stress altered phenotype and function of splenic immune cells. These findings may have implications for the healing of bite wounds that are often associated with social stress in rodents.     

Chronic stress enhances progression of acute lymphoblastic leukemia via β-adrenergic signaling

Clinical studies suggest that stress-related biobehavioral factors can accelerate the progression of hematopoietic cancers such as acute lymphoblastic leukemia (ALL), but it is unclear whether such effects are causal or what biological pathways mediate such effects. Given the network of sympathetic nervous system (SNS) fibers that innervates the bone marrow to regulate normal (non-leukemic) hematopoietic progenitor cells, we tested the possibility that stress-induced SNS signaling might also affect ALL progression. In an orthotopic mouse model, Nalm-6 human pre-B ALL cells were transduced with the luciferase gene for longitudinal bioluminescent imaging and injected i.v. into male SCID mice for bone marrow engraftment. Two weeks of daily restraint stress significantly enhanced ALL tumor burden and dissemination in comparison to controls, and this effect was blocked by the β-adrenergic antagonist, propranolol. Although Nalm-6 ALL cells expressed mRNA for β1- and β3-adrenergic receptors, they showed no evidence of cAMP signaling in response to norepinephrine, and norepinephrine failed to enhance Nalm-6 proliferation in vitro. These results show that chronic stress can accelerate the progression of human pre-B ALL tumor load via a β-adrenergic signaling pathway that likely involves indirect regulation of ALL biology via alterations in the function of other host cell types such as immune cells or the bone marrow microenvironment.     

Physical defeat reduces the sensitivity of murine splenocytes to the suppressive effects of corticosterone

Social disruption (SDR) in male mice reduces the sensitivity of their splenocytes to the actions of glucocorticoids. To determine whether physical defeat is necessary for the development of this reduced sensitivity, a modification of the SDR paradigm was employed in which mice were exposed to fighting conspecifics in the presence or absence of physical contact. This was accomplished by dividing a cage of 5 resident male C57BL/6 mice in half with a wire mesh partition so that 2 of the mice in the cage (SDR Physical Contact mice) fought and were defeated by an aggressive male C57BL/6 intruder that was placed into the cage for 2h for up to 6 days, while the remaining 3 resident mice (SDR Sensory Contact mice) were on the opposite side of the partition and thus prevented from physically interacting with the intruder. Although both the SDR Physical Contact and the SDR Sensory Contact mice had significantly elevated corticosterone levels and displayed submissive postures toward the intruder, only the SDR Physical Contact animals developed functional glucocorticoid resistance. The viability of LPS-stimulated splenocytes cultured from the SDR Physical Contact mice was not affected by pharmacological doses of corticosterone, whereas splenocyte viability was significantly reduced by corticosterone in cultured cells from SDR Sensory Contact and control mice. This study indicates that exposure to a stressful environment in the absence of physical attack does not reduce the sensitivity of murine splenocytes to the suppressive effects of corticosterone.     

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^bNP_366-74CD8⁺ 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.     

Gene expression analysis reveals altered brain transcription of glutamate receptors and inflammatory genes in a patient with chronic focal (Rasmussen's) encephalitis

Following social disruption (SDR) stress in male mice, corticosterone resistance of splenocytes was accompanied by enhanced LPS-stimulated interleukin (IL)-6 secretion. The present study examined the role of IL-6 in the development of corticosterone resistance. Addition of IL-6 to control splenocyte cultures did not induce corticosterone resistance. SDR also elevated IL-6 in plasma and liver, but not in spleen. IL-6 deficient mice that were exposed to SDR developed glucocorticoid resistance despite the absence of systemic IL-6. These findings suggest that although SDR enhanced IL-6 responses, IL-6 was not essential for the development of stress-induced splenocyte corticosterone resistance.     

Differential effects of sympathetic nervous system and hypothalamic–pituitary–adrenal axis on systemic immune cells after severe experimental stroke

Infectious complications are the leading cause of death in the post-acute phase of stroke. Post-stroke immunodeficiency is believed to result from neurohormonal dysregulation of the sympathetic nervous system (SNS) and hypothalamic–pituitary–adrenal (HPA) axis. However, the differential effects of these neuroendocrine systems on the peripheral immune cells are only partially understood. Here, we determined the impact of the hormones of the SNS and HPA on distinct immune cell populations and characterized their interactions after stroke.At various time points after cortical or extensive hemispheric cerebral ischemia, plasma cortisone, corticosterone, metanephrine and adrenocorticotropic hormone (ACTH) levels were measured in mice. Leukocyte subpopulations were flow cytometrically analyzed in spleen and blood. To investigate their differential sensitivity to stress hormones, splenocytes were incubated in vitro with prednisolone, epinephrine and their respective receptor blockers. Glucocorticoid receptor (GCR) and beta2-adrenergic receptor (β2-AR) on leukocyte subpopulations were quantified by flow cytometry. In vivo effects of GCR and selective β2-AR blockade, respectively, were defined on serum hormone concentrations, lymphopenia and interferon-γ production after severe ischemia.We found elevated cortisone, corticosterone and metanephrine levels and associated lymphocytopenia only after extensive brain infarction. Prednisolone resulted in a 5 times higher cell death rate of splenocytes than epinephrine in vitro. Prednisolone and epinephrine-induced leukocyte cell death was prevented by GCR and β2-AR blockade, respectively. In vivo, only GCR blockade prevented post ischemic lymphopenia whereas β2-AR preserved interferon-γ secretion by lymphocytes. GCR blockade increased metanephrine levels in vivo and prednisolone, in turn, decreased β2-AR expression on lymphocytes.In conclusion, mediators of the SNS and the HPA axis differentially affect the systemic immune system after stroke. Moreover, our findings suggest a negative-feedback of corticosteroids on the sympathetic axis which may control the post-stroke stress-reaction. This complex interplay between the HPA and the SNS after stroke has to be considered when targeting the neurohormonal systems in the post acute phase of severe stroke.     

When not enough is too much: the role of insufficient glucocorticoid signaling in the pathophysiology of stress-related disorders

OBJECTIVE: Previous theories have emphasized the role of excessive glucocorticoid activity in the pathology of chronic stress. Nevertheless, insufficient glucocorticoid signaling (resulting from decreased hormone bioavailability or reduced hormone sensitivity) may have equally devastating effects on bodily function. Such effects may be related in part to the role of glucocorticoids in restraining activation of the immune system and other components of the stress response, including the sympathetic nervous system (SNS) and corticotropin-releasing hormone (CRH). METHOD: The literature on neuroendocrine function and glucocorticoid-relevant pathologies in stress-related neuropsychiatric disorders, including posttraumatic stress disorder and major depression, was reviewed. RESULTS: Although not occurring together, both hypocortisolism and reduced responsiveness to glucocorticoids (as determined by dexamethasone challenge tests) were reliably found. Stress-related neuropsychiatric disorders were also associated with immune system activation/inflammation, high SNS tone, and CRH hypersecretion, which are all consistent with insufficient glucocorticoid-mediated regulation of stress hyperresponsiveness. Finally, antidepressants, a mainstay in the treatment of stress-related disorders, were regularly associated with evidence of enhanced glucocorticoid signaling. CONCLUSIONS: Neuroendocrine data provide evidence of insufficient glucocorticoid signaling in stress-related neuropsychiatric disorders. Impaired feedback regulation of relevant stress responses, especially immune activation/inflammation, may, in turn, contribute to stress-related pathology, including alterations in behavior, insulin sensitivity, bone metabolism, and acquired immune responses. From an evolutionary perspective, reduced glucocorticoid signaling, whether achieved at the level of the hormone or its receptor, may foster immune readiness and increase arousal. Emphasis on insufficient glucocorticoid signaling in stress-related pathology encourages development of therapeutic strategies to enhance glucocorticoid signaling pathways.     

Social disruption-induced glucocorticoid resistance: kinetics and site specificity

Social disruption (SDR) of male mice has been shown to induce a state of functional glucocorticoid (GC) resistance in splenocytes. The present study demonstrated that GC resistance developed following repeated, but not acute exposure to SDR. GC resistance was long-lasting and persisted for at least 10 days after stress. In contrast, SDR did not alter cytokine secretion from peritoneal mononuclear cells treated with corticosterone. These findings suggest that SDR-induced GC resistance may be restricted to specific sites such as the spleen.     

Immune alterations induced by social defeat do not alter the course of an on-going BCG infection in mice

The timing for applying stressor and primary immunization is known to influence the nature of the immune alterations induced by stress. The aim of the present study was to investigate the consequences of a stress occurring several days after the beginning of a primary infection on the host resistance. For this purpose, we investigated the effects of repeated social defeat on the immune response of mice infected with BCG 11 days before. In vitro production of cytokines in response to LPS or tuberculin, and the sensitivity of spleen cells to corticosterone were assessed 8 days after the end of the stress. Bacterial growth was assessed in the spleen. We demonstrated that social defeat in BCG-infected mice induced a long-term increase in IL-6 and IL-10 production in response to LPS but did not modify the sensitivity of spleen cells to corticosterone. Stress did not affect the specific response to BCG, as shown by the production of cytokines in tuberculin-stimulated cultures. Accordingly, social defeat was unable to influence the mycobacterial growth in vivo. These results support the hypothesis postulating that stress does not affect antigen-specific response when it is applied after priming.     

Effects of social defeat stress on dopamine D2 receptor isoforms and proteins involved in intracellular trafficking

Background

Chronic social defeat stress induces depression and anxiety-like behaviors in rodents and also responsible for differentiating defeated animals into stress susceptible and resilient groups. The present study investigated the effects of social defeat stress on a variety of behavioral parameters like social behavior, spatial learning and memory and anxiety like behaviors. Additionally, the levels of various dopaminergic markers, including the long and short form of the D2 receptor, and total and phosphorylated dopamine and cyclic adenosine 3′,5′-monophosphate regulated phosphoprotein-32, and proteins involved in intracellular trafficking were assessed in several key brain regions in young adult mice.

Methods

Mouse model of chronic social defeat was established by resident-intruder paradigm, and to evaluate the effect of chronic social defeat, mice were subjected to behavioral tests like spontaneous locomotor activity, elevated plus maze (EPM), social interaction and Morris water maze tests.

Results

Mice were divided into susceptible and unsusceptible groups after 10 days of social defeat stress. The susceptible group exhibited greater decreases in time spent in the open and closed arms compared to the control group on the EPM. In the social interaction test, the susceptible group showed greater increases in submissive and neutral behaviors and greater decreases in social behaviors relative to baseline compared to the control group. Furthermore, increased expression of D2L, D2S, Rab4, and G protein-coupled receptor associated sorting protein-1 was observed in the amygdala of the susceptible group compared to the control group.

Conclusion

These findings suggest that social defeat stress induce anxiety-like and altered social interacting behaviors, and changes in dopaminergic markers and intracellular trafficking-related proteins.