Chronic psychosocial stress has been implicated in the etiology and progression of psychiatric disorders such as major depression and post-traumatic stress disorder (PTSD). In mice, we study the effects of psychosocial stress in a conflict paradigm that leads to behavioral alterations such as deficits in social interaction or increases in anhedonia, anxiety, and depressive-like states. In our social defeat paradigm, two mice are placed in a continuous dyadic living relationship in which the subordinate experimental mouse is chronically exposed to and periodically defeated by a dominant mouse. Over the course of weeks in this living situation, the experimental mouse develops anxious and depressive-like behaviors that are associated with distinct neurochemical alterations in identified brain circuits. A decreased rate of hippocampal neurogenesis is one well-documented readout of stress effects. Thus the paradigm is an ethologically relevant and validated model that can provide insights into the bases for mental illness in humans. Our research focuses on the roles played by immune factors in the etiology of psychosocial stress-induced depressive states. We hypothesize that there is a bidirectional dialog between the brain and the immune system that serves to maintain homeostasis in healthy states, but disturbances within this dialog lead to homeostatic corrections or dysfunction that contribute to the onset and course of psychiatric disease. Therefore, we measure immune system parameters as we manipulate psychosocial processes that affect brain states. Two projects investigate immune changes in rodents undergoing psychological stress; one focuses on the role of the adaptive immune system in maintaining homeostatic balance, and the second investigates stress-induced changes in immune cells and molecules within the brain itself. 1) We conducted experiments aimed at addressing the role of the adaptive immune system in controlling mood states in the chronic social defeat (CSD) model. The adaptive immune system, separate from the innate immune system, comprises cells lymphocytes that adapt, i.e., acquire and retain a memory of prior challenges. Surprisingly, lymphocytes appear to exert effects on the brain and behavior as suggested by results of lymphocyte transfer experiments. We developed a novel approach to the question by studying the Rag2-/- mouse, which lacks a gene needed to produce mature lymphocytes. We adoptively transferred lymphocytes into this lymphopenic mouse from normal donor mice that had either been chronically stressed or unstressed, and after two weeks of lymphocyte reconstitution in the host mouse, behavioral tests and hippocampal cell proliferation assays were performed. The mice that received cells from defeated mice showed anti-depressive and anxiolytic behaviors relative to mice that received no cell transfer or transfer of cells from home-cage control mice. These surprising findings suggested that the adaptive immune system retains a memory for adverse events and attempts to return the host animal to a condition of homeostasis. Current work examines the molecular and cellular determinants of the interaction and the anatomical and humoral pathways by which the immune system affects brain function and structure. We are particularly focused on the meninges as a site of interaction between the periphery and the brain. The meninges are a special component of the blood-brain barrier (BBB) because immune cells only very sparsely enter the brain itself but do reside in the subarachnoid spaces of the meninges where they can release bioactive molecules that circulate in the interstitial cerebrospinal fluid to widely influence brain activity. Whole-mount meningeal preparations and flow cytometry of cells residing in the meninges are used to analyze lymphocytes in this compartment. We characterize activation states and gene expression profiles in these cells. The profiles guide us to program lymphocytes in cell culture to display similar properties. The goal is to introduce cultured cells into an animal with dysregulated affect to confer antidepressant properties. Such studies may lead to insights into new targets for therapeutic interventions in psychiatric disorders. 2) The immune system comprises cells of peripheral immune organs and also specialized immune cells within the brain microglia that share many properties with peripheral myeloid-derived cells. Our recent research showed that CSD triggered a CNS immune response within emotion-processing brain areas, which may be one basis for our finding that chronically stressed mice had reduced levels of myelination in the prefrontal cortex. We have data showing effects of acute and chronic social defeat stress on microglial proliferation and activation states in the prefrontal cortex. These activation states can affect glial and neuronal function, which may differentially direct behavioral outcomes associated with either susceptibility or resilience to the effects of the stress. We have analyzed microglia isolated by flow cytometry to characterize cell-surface markers of activation and by microarray to assess gene expression patterns. The microarray data indicated that microglia isolated from defeated mice that had been susceptible to the depressive effects of social defeat were functionally distinct from microglia taken from defeated mice that showed behavioral resilience to the stress procedure. The microglial gene expression characteristics of the resilient group were more like those from unstressed control mice, suggesting that microglial activity contributes to the brain states that afford resilience to the effects of psychosocial stress. Gene expression profiles in the microglia from susceptible mice showed evidence of inflammation, phagocytosis, extracellular matrix breakdown, oxidative stress, and extravasation. These findings were assayed by histochemical and ex vivo assay methods. In the susceptible mice only, local breeches of the BBB and brain micro-bleeds were found. These were associated with microglial immunostaining of matrix metalloproteinases MMP-8 and MMP-9, which indicate degradation of the extracellular matrix. Susceptible mice also showed elevated levels of hydidroethidium (DHE) in cells, indicating increased levels of oxidative stress. Drugs that prevent the conversion of microglia to a reactive pro-inflammatory state seen in the susceptible group may protect the animals from the depressive effects of the defeat stress. In one experiment, drugs that block matrix metalloproteinase activity were effective, In another experiment, a reactive oxygen species (ROS) inhibitor N-acetyl cysteine was effective. Studies are underway to explore the role played by microglia in these effects by depleting microglia using the CSF-1R antagonist drug PLX5622.
The aims of the work are to demonstrate a causal relationship between stress-induced immune alterations and susceptibility or resilience to the stress, and to suggest new therapeutic targets for the treatment of stress-related mood disorders.
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