Deciphering sex differences in the immune system and depression.

Certain mood disorders and autoimmune diseases are predominately female diseases but we do not know why. Here, we explore the relationship between depression and the immune system from a sex-based perspective. This review characterizes sex differences in the immune system in health and disease. We explore the contribution of gonadal and stress hormones to immune function at the cellular and molecular level in the brain and body. We propose hormonal and genetic sex specific immune mechanisms that may contribute to the etiology of mood disorders.

Actions of Steroids: New Neurotransmitters.

Over the past two decades, the classical understanding of steroid action has been updated to include rapid, membrane-initiated, neurotransmitter-like functions. While steroids were known to function on very short time spans to induce physiological and behavioral changes, the mechanisms by which these changes occur are now becoming more clear. In avian systems, rapid estradiol effects can be mediated via local alterations in aromatase activity, which precisely regulates the temporal and spatial availability of estrogens. Acute regulation of brain-derived estrogens has been shown to rapidly affect sensorimotor function and sexual motivation in birds. In rodents, estrogens and progesterone are critical for reproduction, including preovulatory events and female sexual receptivity. Membrane progesterone receptor as well as classical progesterone receptor trafficked to the membrane mediate reproductive-related hypothalamic physiology, via second messenger systems with dopamine-induced cell signals. In addition to these relatively rapid actions, estrogen membrane-initiated signaling elicits changes in morphology. In the arcuate nucleus of the hypothalamus, these changes are needed for lordosis behavior. Recent evidence also demonstrates that membrane glucocorticoid receptor is present in numerous cell types and species, including mammals. Further, membrane glucocorticoid receptor influences glucocorticoid receptor translocation to the nucleus effecting transcriptional activity. The studies presented here underscore the evidence that steroids behave like neurotransmitters to regulate CNS functions. In the future, we hope to fully characterize steroid receptor-specific functions in the brain.

Chronic variable stress leads to sex specific gut microbiome alterations in mice.

Stress has been implicated in the incidence and severity of psychiatric and gastrointestinal disorders. The immune system is capable of modulating the activity and composition of the gut following stress and vice versa. In this study we sought to examine the sequential relationship between immune signaling and microbiome composition occurring in male and female mice over time using a variable stress paradigm. Tissue was collected prior to, during, and after the stress paradigm from the same mice. Cytokines from plasma and brain were quantified using a multiplexed cytokine assay. Fecal samples were collected at the same timepoints and 16S rRNA amplicon sequencing was performed to determine the relative abundance of microbiota residing in the guts of stressed and control mice. We found sex differences in the response of the gut microbiota to stress following 28 days of chronic variable stress but not 6 days of sub-chronic variable stress. Immune activation was quantified in the nucleus accumbens immediately following Sub-chronic variable when alterations of gut composition had not yet occurred. In both sexes, 28 days of stress induced significant changes in the proportion of Erysipelotrichaceae and Lactobacillaceae, but in opposite directions for male and female mice. Alterations to the gut microbiome in both sexes were associated with changes in cytokines related to eosinophilic immune activity. Our use of an animal stress model reveals the immune mechanisms that may underly changes in gut microbiome composition during and after stress. This study reveals potential drug targets and microbiota of interest for the intervention of stress related conditions.

Translating the Transcriptome: Sex Differences in the Mechanisms of Depression and Stress, Revisited.

The past decade has produced a plethora of studies examining sex differences in the transcriptional profiles of stress and mood disorders. As we move forward from accepting the existence of extensive molecular sex differences in the brain to exploring the purpose of these sex differences, our approach must become more systemic and less reductionist. Earlier studies have examined specific brain regions and/or cell types. To use this knowledge to develop the next generation of personalized medicine, we need to comprehend how transcriptional changes across the brain and/or the body relate to each other. We provide an overview of the relationships between baseline and depression/stress-related transcriptional sex differences and explore contributions of preclinically identified mechanisms and their impacts on behavior.

Testing the Limits of Sex Differences Using Variable Stress.

Depression is a chronic disease that affects nearly twice as many women as men, and symptoms can differ by sex. Preclinical models disproportionately use male subjects and test a single behavioral endpoint immediately at the cessation of stress. We conducted variable stress in male and female mice for 6, 28, and 56 days, and measured behavior with a battery chosen to match research domain criteria. To examine individual differences, we generated a composite z score to measure stress susceptibility across behavioral tests. We also tested behavior following a 30-day recovery period to evaluate the duration of the stress effects. Females, but not males, were susceptible to 6 days of variable stress when behavioral testing started 24 h later. If behavioral testing was conducted 30 days later both males and females expressed stress related behaviors. Males and females were stress susceptible to 28 days of variable stress and effects were long lasting. Both sexes habituated to 56 days of variable stress, but anxiety associated measures still showed persistence. Performance on specific behavioral tests was often different between individuals and between sexes, and not all stressed animals were susceptible to all tested behaviors. These studies confirm that behavioral sex differences are detected in response to variable stress, and reveal information about individual differences. Use of a test battery that measures varying endophenotypes can be combined into a single stress susceptibility score as a tool similar to the scales/inventories used for the study of depression in humans. We present these data with the goal of furthering the field's understanding sex differences and how they shape the biology of mood disorders.

Sexually dimorphic neuroimmune response to chronic opioid treatment and withdrawal.

Opioid use disorder is a leading cause of morbidity and mortality in the United States. Increasing pre-clinical and clinical evidence demonstrates sex differences in opioid use and dependence. However, the underlying molecular mechanisms contributing to these effects, including neuroinflammation, are still obscure. Therefore, in this study, we investigated the effect of oxycodone exposure and withdrawal on sex- and region-specific neuroimmune response. Real-time PCR and multiplex cytokine array analysis demonstrated elevated neuroinflammation with increased pro-inflammatory cytokine levels, and aberrant oligodendroglial response in reward neurocircuitry, following withdrawal from chronic oxycodone treatment. Chronic oxycodone and withdrawal treated male mice had lower mRNA expression of TMEM119 along with elevated protein levels of pro-inflammatory cytokines/chemokines and growth factors (IL-1ß, IL-2, IL-7, IL-9, IL-12, IL-15, IL17, M-CSF, VEGF) in the prefrontal cortex (PFC) as compared to their female counterparts. In contrast, reduced levels of pro-inflammatory cytokines/chemokines (IL-1ß, IL-6, IL-9, IL-12, CCL11) was observed in the nucleus accumbens (NAc) of oxycodone and withdrawal-treated males as compared to female mice. No treatment specific effects were observed on the mRNA expression of putative microglial activation markers (Iba1, CD68), but an overall sex specific decrease in the mRNA expression of Iba1 and CD68 was found in the PFC and NAc of male mice as compared to females. Moreover, a sex and region-specific increase in the mRNA levels of oligodendrocyte lineage markers (NG2, Sox10) was also observed in oxycodone and withdrawal treated animals. These findings may open a new avenue for the development of sex-specific precision therapeutics for opioid dependence by targeting region-specific neuroimmune signaling.

Sex and region-specific effects of variable stress on microglia morphology.

Major Depressive Disorder (MDD) is a common and debilitating mood disorder that is more prevalent in women than men. In humans, PET imaging of microglia activation is currently being explored as a potential biomarker of MDD and suicidal ideation. Stress is a trigger for many mood disorders, including MDD. Microglial changes in morphology and activation state in response to stress has been reported in various brain regions, but most studies only examined male subjects. Here we report changes in microglia morphology in the nucleus accumbens (NAc) and subregions of the hippocampus (HPC) in both male and female mice following variable stress of 6 or 28 days in duration. Our data demonstrate that after 6 days of stress, microglia in the female NAc and dentate gyrus have a reduction in homeostatic associated morphology and an increase in primed microglia. After 28 days some of these sex specific stress effects were still present in microglia within the NAc but not the dentate gyrus. There were no effects of stress in either sex at either timepoint in CA1. In female mice, anti-inflammatory activation of microglia using rosiglitazone promoted sociability behavior after 6 days of stress. Furthermore, both drug and stress have impact on microglia morphology and activation state in the NAc. These data suggest that microglia morphology and activation state are altered by 6 days of variable stress in a region-specific manner and may contribute to, or potentially compensate for, the onset of stress susceptibility rather than impacting long term exposure to stress.

Inflaming sex differences in mood disorders.

Men and women often experience different symptoms or rates of occurrence for a variety of mood disorders. Many of the symptoms of mood disorders overlap with autoimmune disorders, which also have a higher prevalence in women. There is a growing interest in exploring the immune system to provide biomarkers for diagnosis of mood disorders, along with new targets for developing treatments. This review examines known sex differences in the immune system and their relationship to mood disorders. We focus on immune alterations associated with unipolar depression, bipolar depression, and anxiety disorders. We describe work from both basic and clinical research examining potential immune mechanisms thought to contribute to stress susceptibility and associated mood disorders. We propose that sex and age are important, intertwined factors that need to be included in future experimental designs if we are going to harness the power of the immune system to develop a new wave of treatments for mood disorders.

Purity and stability of the membrane-limited glucocorticoid receptor agonist dexamethasone-BSA.

Cellular effects of glucocorticoids can be separated into classical transcriptional regulation via activation of the canonical nuclear glucocorticoid receptor and rapid actions mediated by activation of one or more putative membrane-associated glucocorticoid receptors that regulate both transcriptional and non-transcriptional signaling. Dexamethasone-bovine serum albumin (Dex-BSA) is one of several membrane-limited steroid receptor agonists. Dex-BSA and other steroid conjugates such as corticosterone-, estradiol- and testosterone-BSA have been used to study rapid steroid effects initiated by putative membrane receptors. The purity and stability of the steroid-BSA conjugate is crucial, therefore, since any steroid that is not bound to or that dissociates from the BSA conjugate could penetrate into the intracellular compartment and confound the experiment. We used fluorine NMR to determine if free Dex could be detected in a commercially available Dex-BSA dissolved in H2O. Non-covalently bound Dex was detected in the Dex-BSA solution, but the level of free Dex remained constant over time and with increasing temperature, indicating that the free Dex was not a result of instability of the Dex-BSA conjugate. The free Dex was lost when the Dex-BSA was denatured and subjected to dialysis, which suggested that it was trapped in the Dex-BSA three-dimensional structure and not covalently bound to the BSA. The purified, renatured Dex-BSA retained its rapid activity, which confirmed that the observed effects of Dex-BSA are not caused by non-covalently-bound Dex. Therefore, the Dex contaminant found in the Dex-BSA solution is likely to be tightly, but non-covalently, bound to BSA, and the Dex-BSA activity remains membrane-limited. Our findings indicate that Dex-BSA remains a suitable membrane-restricted glucocorticoid receptor agonist, but suggest that denaturing purification is a useful control for the study of membrane-initiated steroid-BSA actions.

Membrane-initiated nuclear trafficking of the glucocorticoid receptor in hypothalamic neurons.

Glucocorticoid binding to the intracellular glucocorticoid receptor (GR) stimulates the translocation of the GR from the cytosol to the nucleus, which leads to the transactivation or transrepression of gene transcription. However, multiple lines of evidence suggest that glucocorticoid signaling can also be initiated from the plasma membrane. Here, we provide evidence for membrane-initiated glucocorticoid signaling by a membrane-impermeant dexamethasone-bovine serum albumin (Dex-BSA) conjugate, which induced GR nuclear trafficking in hypothalamic neurons in vitro and in vivo. The GR nuclear translocation induced by a membrane-impermeant glucocorticoid suggests trafficking of an unliganded GR. The membrane-initiated GR trafficking was not blocked by inhibiting ERK MAPK, p38 MAPK, PKA, Akt, Src kinase, or calcium signaling, but was inhibited by Akt activation. Short-term exposure of hypothalamic neurons to dexamethasone (Dex) activated the glucocorticoid response element (GRE), suggesting transcriptional transactivation, whereas exposure to the Dex-BSA conjugate failed to activate the GRE, suggesting differential transcriptional activity of the liganded compared to the unliganded GR. Microarray analysis revealed divergent transcriptional regulation by Dex-BSA compared to Dex. Together, our data suggest that signaling from a putative membrane glucocorticoid receptor induces the trafficking of unliganded GR to the nucleus, which elicits a pattern of gene transcription that differs from that of the liganded receptor. The differential transcriptional signaling by liganded and unliganded receptors may contribute to the broad range of genetic regulation by glucocorticoids, and may help explain some of the different off-target actions of glucocorticoid drugs.

Further evidence for a membrane receptor that binds glucocorticoids in the rodent hypothalamus.

In parallel with their well-characterized delayed genomic effects, steroid hormones exhibit rapid, non-genomic effects at molecular, cellular and behavioral levels. We have proposed a model of rapid, non-genomic glucocorticoid inhibition of hypothalamic neuroendocrine cells through a putative membrane-associated glucocorticoid receptor (GR). Here we tested for plasma membrane GR immunoreactivity and binding in the hypothalamic supraoptic and paraventricular nuclei. Selective cross-linking of membrane proteins with membrane-impermeant BS3 and subsequent Western blot analysis with a monoclonal GR antibody revealed a reduction in the intensities of a ∼98kDa immunoreactive band and a ∼64kDa band in the rat paraventricular and supraoptic nuclei, and of a 64kDa band in hippocampal tissue, which suggested that these proteins are associated with the membrane. Saturation binding of [3H]-corticosterone and [3H]-dexamethasone in rat and mouse hypothalamic tissue revealed a Kd 4-24-fold lower and a Bmax 4-7-fold lower for the membrane-associated GR compared to the intracellular GR, suggesting a lower affinity and abundance of the glucocorticoid binding sites in the membrane than in the cytosol. Together, these findings suggest the presence of a low-affinity, low-abundance membrane-associated GR in the hypothalamus that shares homology with the intracellular GR, and are consistent with physiological evidence of rapid, non-genomic glucocorticoid actions in hypothalamic neuroendocrine cells that are GR dependent.

Endocannabinoid Regulation of Neuroendocrine Systems.

The hypothalamus is a part of the brain that is critical for sustaining life through its homeostatic control and integrative regulation of the autonomic nervous system and neuroendocrine systems. Neuroendocrine function in mammals is mediated mainly through the control of pituitary hormone secretion by diverse neuroendocrine cell groups in the hypothalamus. Cannabinoid receptors are expressed throughout the hypothalamus, and endocannabinoids have been found to exert pronounced regulatory effects on neuroendocrine function via modulation of the outputs of several neuroendocrine systems. Here, we review the physiological regulation of neuroendocrine function by endocannabinoids, focusing on the role of endocannabinoids in the neuroendocrine regulation of the stress response, food intake, fluid homeostasis, and reproductive function. Cannabis sativa (marijuana) has a long history of recreational and/or medicinal use dating back to ancient times. It was used as an analgesic, anesthetic, and antianxiety herb as early as 2600 B.C. The hedonic, anxiolytic, and mood-elevating properties of cannabis have also been cited in ancient records from different cultures. However, it was not until 1964 that the psychoactive constituent of cannabis, Δ(9)-tetrahydrocannabinol, was isolated and its chemical structure determined (Gaoni & Mechoulam, 1964).