The role of microglia in 67NR mammary tumor-induced suppression of brain responses to immune challenges in female mice.

It is poorly understood how solid peripheral tumors affect brain neuroimmune responses despite the various brain-mediated side effects and higher rates of infection reported in cancer patients. We hypothesized that chronic low-grade peripheral tumor-induced inflammation conditions microglia to drive suppression of neuroinflammatory responses to a subsequent peripheral immune challenge. Here, Balb/c murine mammary tumors attenuated the microglial inflammatory gene expression responses to lipopolysaccharide (LPS) and live Escherichia coli (E. coli) challenges and the fatigue response to an E. coli infection. In contrast, the inflammatory gene expression in response to LPS or a toll-like receptor 2 agonist of Percoll-enriched primary microglia cultures was comparable between tumor-bearing and -free mice, as were the neuroinflammatory and sickness behavioral responses to an intracerebroventricular interleukin (IL)-1ß injection. These data led to the hypothesis that Balb/c mammary tumors blunt the neuroinflammatory responses to an immune challenge via a mechanism involving tumor suppression of the peripheral humoral response. Balb/c mammary tumors modestly attenuated select circulating cytokine responses to LPS and E. coli challenges. Further, a second mammary tumor/mouse strain model (E0771 tumors in C57Bl/6 mice) displayed mildly elevated inflammatory responses to an immune challenge. Taken together, these data indicate that tumor-induced suppression of neuroinflammation and sickness behaviors may be driven by a blunted microglial phenotype, partly because of an attenuated peripheral signal to the brain, which may contribute to infection responses and behavioral side effects reported in cancer patients. Finally, these neuroimmune effects likely vary based on tumor type and/or host immune phenotype.

Oral Sciences PhD Program Enrollment, Graduates, and Placement: 1994 to 2016.

For decades, dental schools in the United States have endured a significant faculty shortage. Studies have determined that the top 2 sources of dental faculty are advanced education programs and private practice. Those who have completed both DDS and PhD training are considered prime candidates for dental faculty positions. However, there is no national database to track those trainees and no evidence to indicate that they entered academia upon graduation. The objective of this study was to assess outcomes of dental school-affiliated oral sciences PhD program enrollment, graduates, and placement between 1994 and 2016. Using the American Dental Association annual survey of advanced dental education programs not accredited by the Commission on Dental Accreditation and data obtained from 22 oral sciences PhD programs, we assessed student demographics, enrollment, graduation, and placement. Based on the data provided by program directors, the average new enrollment was 33, and graduation was 26 per year. A total of 605 graduated; 39 did not complete; and 168 were still in training. Among those 605 graduates, 211 were faculty in U.S. academic institutions, and 77 were faculty in foreign institutions. Given that vacant budgeted full-time faculty positions averaged 257 per year during this period, graduates from those oral sciences PhD programs who entered academia in the United States would have filled 9 (3.6%) vacant faculty positions per year. Therefore, PhD programs have consistently generated only a small pipeline of dental school faculty. Better mentoring to retain talent in academia is necessary. Stronger support and creative funding plans are essential to sustain the PhD program. Furthermore, the oral sciences PhD program database should be established and maintained by dental professional organizations to allow assessments of training models, trends of enrollment, graduation, and placement outcomes.

Microglial recruitment of IL-1ß-producing monocytes to brain endothelium causes stress-induced anxiety.

Psychosocial stress contributes to the development of anxiety and depression. Recent clinical studies have reported increased inflammatory leukocytes in circulation of individuals with stress-related psychiatric disorders. Parallel to this, our work in mice shows that social stress causes release of inflammatory monocytes into circulation. In addition, social stress caused the development of prolonged anxiety that was dependent on inflammatory monocytes in the brain. Therefore, we hypothesize that chronic stress drives the production of inflammatory monocytes that are actively recruited to the brain by microglia, and these monocytes augment neuroinflammatory signaling and prolong anxiety. Here we show that repeated social defeat stress in mice activated threat appraisal centers in the brain that spatially coincided with microglial activation and endothelial facilitation of monocyte recruitment. Moreover, microglial depletion with a CSF1R antagonist prior to stress prevented the recruitment of monocytes to the brain and abrogated the development of anxiety. Cell-specific transcriptional profiling revealed that microglia selectively enhanced CCL2 expression, while monocytes expressed the pro-inflammatory cytokine interleukin-1ß (IL-1ß). Consistent with these profiles, the recruited inflammatory monocytes with stress adhered to IL-1R1+ neurovascular endothelial cells and this interaction was blocked by microglial depletion. Furthermore, disruption of IL-1ß signaling by caspase-1KO specifically within bone marrow-derived cells revealed that monocytes promoted anxiogenesis through stimulation of neurovascular IL-1R1 by IL-1ß. Collectively, the development of anxiety during stress was caused by microglial recruitment of IL-1ß-producing monocytes, which stimulated brain endothelial IL-1R1. Thus, monocyte IL-1ß production represents a novel mechanism that underlies behavioral complications associated with stress-related psychiatric disorders.

Peripheral and central effects of repeated social defeat stress: monocyte trafficking, microglial activation, and anxiety.

The development and exacerbation of depression and anxiety are associated with exposure to repeated psychosocial stress. Stress is known to affect the bidirectional communication between the nervous and immune systems leading to elevated levels of stress mediators including glucocorticoids (GCs) and catecholamines and increased trafficking of proinflammatory immune cells. Animal models, like the repeated social defeat (RSD) paradigm, were developed to explore this connection between stress and affective disorders. RSD induces activation of the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis activation, increases bone marrow production and egress of primed, GC-insensitive monocytes, and stimulates the trafficking of these cells to tissues including the spleen, lung, and brain. Recently, the observation that these monocytes have the ability to traffic to the brain perivascular spaces and parenchyma have provided mechanisms by which these peripheral cells may contribute to the prolonged anxiety-like behavior associated with RSD. The data that have been amassed from the RSD paradigm and others recapitulate many of the behavioral and immunological phenotypes associated with human anxiety disorders and may serve to elucidate potential avenues of treatment for these disorders. Here, we will discuss novel and key data that will present an overview of the neuroendocrine, immunological and behavioral responses to social stressors.

Social defeat promotes a reactive endothelium in a brain region-dependent manner with increased expression of key adhesion molecules, selectins and chemokines associated with the recruitment of myeloid cells to the brain.

Repeated social defeat (RSD) in mice causes myeloid cell trafficking to the brain that contributes to the development of prolonged anxiety-like behavior. Myeloid cell recruitment following RSD occurs in regions where neuronal and microglia activation is observed. Thus, we hypothesized that crosstalk between neurons, microglia, and endothelial cells contributes to brain myeloid cell trafficking via chemokine signaling and vascular adhesion molecules. Here we show that social defeat caused an exposure- and brain region-dependent increase in several key adhesion molecules and chemokines involved in the recruitment of myeloid cells. For example, RSD induced distinct patterns of adhesion molecule expression that may explain brain region-dependent myeloid cell trafficking. VCAM-1 and ICAM-1 mRNA expression were increased in an exposure-dependent manner. Furthermore, RSD-induced VCAM-1 and ICAM-1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Next, mRNA expression of additional adhesion molecules (E- and P-selectin, PECAM-1) and chemokines (CXCL1, CXCL2, CXCL12, CCL2) were determined in the brain. Social defeat induced an exposure-dependent increase in mRNA levels of E-selectin, CXCL1, and CXCL2 that increased with additional days of social defeat. While CXCL12 was unaffected by RSD, CCL2 expression was increased by six days of social defeat. Last, comparison between enriched CD11b(+) cells (microglia/macrophages) and enriched GLAST-1(+)/CD11b(-) cells (astrocytes) revealed RSD increased mRNA expression of IL-1ß, CCL2, and CXCL2 in microglia/macrophages but not in astrocytes. Collectively, these data indicate that key mediators of leukocyte recruitment were increased in the brain vasculature following RSD in an exposure- and brain region-dependent manner.

Psychosocial stress and inflammation in cancer.

Stress-induced immune dysregulation results in significant health consequences for immune related disorders including viral infections, chronic autoimmune disease, and tumor growth and metastasis. In this mini-review we discuss the sympathetic, neuroendocrine and immunologic mechanisms by which psychosocial stress can impact cancer biology. Both human and animal studies have shown the sympathetic and neuroendocrine responses to psychosocial stress significantly impacts cancer, in part, through regulation of inflammatory mediators. Psychosocial stressors stimulate neuroendocrine, sympathetic, and immune responses that result in the activation of the hypothalamic-pituitary-adrenal (HPA)-axis, sympathetic nervous system (SNS), and the subsequent regulation of inflammatory responses by immune cells. Social disruption (SDR) stress, a murine model of psychosocial stress and repeated social defeat, provides a novel and powerful tool to probe the mechanisms leading to stress-induced alterations in inflammation, tumor growth, progression, and metastasis. In this review, we will focus on SDR as an important model of psychosocial stress in understanding neural-immune mechanisms in cancer.

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 ß-adrenergic receptors (ßARs) 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 ß-adrenergic antagonist propranolol. Pre-treatment with the ß-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.

Repeated social stress enhances the innate immune response to a primary HSV-1 infection in the cornea and trigeminal ganglia of Balb/c mice.

Three to 5 days after a primary HSV-1 infection, macrophages infiltrate into the trigeminal ganglia (TG) and produce anti-viral cytokines to reduce viral replication. Previous research demonstrated that social disruption stress (SDR) enhances the trafficking of monocytes/macrophages from the bone marrow to the spleen and increases pro-inflammatory cytokine production in vitro and in vivo. The impact of SDR on the trafficking of these cells to loci of herpes simplex virus type 1 (HSV-1) infection and subsequent function has not been examined. The following studies were designed to determine whether SDR would enhance the innate immune response during a primary HSV-1 infection by increasing the number of macrophages in the cornea and TG, thus increasing anti-viral cytokine production and reducing viral replication. BALB/c mice were exposed to six cycles of SDR prior to ocular infection with HSV-1 McKrae virus. Flow cytometric analysis of cells from the TG revealed an increase in the percentage of CD11b+ macrophages in SDR mice compared to controls. Immune cell infiltration into the cornea, however, could not be determined due to low cell numbers. Although gene expression of IFN-beta was decreased, SDR increased gene expression of IFN-alpha, and TNF-alpha, in the cornea and TG. Examination of viral proteins showed decreased expression of infected cell protein 0 (ICP0), glycoprotein B (gB), glycoprotein H (gH) and latency-associated transcript (LAT) in the TG, however, expression of ICP0 and gB were elevated in the cornea of SDR mice. These results indicate that the innate immune response to HSV-1 was altered and enhanced by the experience of repeated social defeat.

Influenza virus infection induces metallothionein gene expression in the mouse liver and lung by overlapping but distinct molecular mechanisms.

Metallothionein I (MT-I) and MT-II have been implicated in the protection of cells against reactive oxygen species (ROS), heavy metals, and a variety of pathological and environmental stressors. Here, we show a robust increase in MT-I/MT-II mRNA level and MT proteins in the livers and lungs of C57BL/6 mice exposed to the influenza A/PR8 virus that infects the upper respiratory tract and lungs. Interleukin-6 (IL-6) had a pronounced effect on the induction of these genes in the liver but not the lung. Treatment of the animals with RU-486, a glucocorticoid receptor antagonist, inhibited induction of MT-I/MT-II in both liver and lung, revealing a direct role of glucocorticoid that is increased upon infection in this induction process. In vivo genomic footprinting (IVGF) analysis demonstrated involvement of almost all metal response elements, major late transcription factor/antioxidant response element (MLTF/ARE), the STAT3 binding site on the MT-I upstream promoter, and the glucocorticoid responsive element (GRE1), located upstream of the MT-II gene, in the induction process in the liver and lung. In the lung, inducible footprinting was also identified at a unique gamma interferon (IFN-gamma) response element (gamma-IRE) and at Sp1 sites. The mobility shift analysis showed activation of STAT3 and the glucocorticoid receptor in the liver and lung nuclear extracts, which was consistent with the IVGF data. Analysis of the newly synthesized mRNA for cytokines in the infected lung by real-time PCR showed a robust increase in the levels of IL-10 and IFN-gamma mRNA that can activate STAT3 and STAT1, respectively. A STAT1-containing complex that binds to the gamma-IRE in vitro was activated in the infected lung. No major change in MLTF/ARE DNA binding activity in the liver and lung occurred after infection. These results have demonstrated that MT-I and MT-II can be induced robustly in the liver and lung following experimental influenza virus infection by overlapping but distinct molecular mechanisms.

Stress and influenza viral infection: modulation of proinflammatory cytokine responses in the lung.

Viral infection of the respiratory tract induces a complex series of cellular and molecular events leading to immunological responses designed to terminate viral replication. Anti-viral immunity involves natural resistance mechanisms that overlap and modulate the development of the subsequent adaptive immune responses. An experimental murine infection with influenza A/PR8 virus was used to examine the effects of stress-induced activation of the nervous and endocrine systems on components of innate immunity. Proinflammatory cytokine responses (IL-1alpha, IL-6 and TNFalpha) were measured in the lungs during an influenza A/PR8 viral infection. For activation of the nervous and endocrine systems, restraint stress (RST) was applied prior to and during infection. Following infection, IL-1alpha increased transiently, while elevated IL-6 persisted; TNFalpha was not detected. RST suppressed virally-induced IL-1alpha, while IL-6 was unaffected. These data demonstrate differential regulation of proinflammatory cytokines by stress. The mechanism underlying suppression of the lung IL-1alpha in stressed mice is currently unknown; its downregulation may contribute to increased viral pathogenesis in stressed individuals.

Evidence for a shift in the Th-1 to Th-2 cytokine response associated with chronic stress and aging.

BACKGROUND: A number of studies have shown that the chronic stress of caring for persons with dementia can have significant immunological consequences as demonstrated by the down-regulation/dysregulation of the cellular immune response. METHODS: Utilizing flow cytometry to measure the percentages and absolute numbers of CD-4(+) and CD-8(+) T lymphocytes producing the cytokines indicative of Th-1, Tc1 and Th-2, and Tc2 cells, we compared spousal caregivers and control subjects. The expression of interleukin-2 (IL-2), interferon gamma (IFN-gamma), and interleukin-10 (IL-10) in the cytoplasm of CD-4(+) and CD-8(+) lymphocytes was assessed. RESULTS: Neither stress nor age was significantly related to the percentage or number of IFNgamma(+)/CD-8(+), IL-2(+)/CD-8(+) cells, or IFNgamma(+), IL-2(+), CD-4(+) cells. However, the percentage of IL-10(+) cells was higher in lymphocytes obtained from caregivers than control subjects. In addition, the significant interaction between stress and aging for IL-10(+)/CD-4(+) and IL-10(+)/CD-8(+) cells demonstrated that the difference between caregivers and control subjects was age dependent; the difference between caregivers and control subjects was substantially larger in younger individuals than in older individuals. CONCLUSIONS: The data are consistent with previous reports on acute stress and suggest that there may also be a shift from a Th-1 to a Th-2 response associated with a chronic stressor such as caregiving. This shift could have implications for an individual's responses to pathogens.

Social stress induces glucocorticoid resistance in macrophages.

Stress-induced levels of plasma glucocorticoid hormones are known to modulate leukocyte function. These experiments examined the effects of a social stressor on the responsiveness of peripheral immune cells. Male mice experienced six evening cycles of social disruption (SDR), in which an aggressive male intruder was placed into their home cage for 2 h. Although circulating corticosterone was elevated in SDR mice, they had enlarged spleens and increased numbers of splenic leukocytes. Splenocytes from SDR and control mice were cultured with lipopolysaccharide and corticosterone. Cells from SDR mice exhibited decreased sensitivity to the antiproliferative effects of corticosterone, suggesting that the peripheral immune cells were resistant to glucocorticoids. In addition, SDR cells produced more interleukin (IL)-6. To determine which cell population was affected, we used antibody-labeled magnetic beads to deplete splenocyte suspensions of B cells or macrophages. Depletion of macrophages from SDR cultures, but not depletion of B cells, abolished both the corticosterone resistance and enhanced IL-6 secretion. These findings demonstrate that a psychosocial stressor induced glucocorticoid resistance in mouse splenic macrophages.

Social stress induces glucocorticoid resistance in subordinate animals.

Introducing an aggressive intruder into a cage of mice (social disruption, SDR) resulted in intense fighting and defeat of the cage residents. Defeat was accompanied by elevated levels of serum corticosterone and nerve growth factor (NGF). Repeated exposure to an intruder induced a state of glucocorticoid resistance in peripheral immune cells. The present study sought to examine the behavioral factors that mediated the development of glucocorticoid resistance following SDR. Glucocorticoid resistance developed in animals that exhibited a subordinate behavioral profile, which consisted of a low tendency for social investigation and a high level of submissive behavior in response to the intruder's attacks. Glucocorticoid resistance was also linked to the presence of injuries due to fighting, but not to changes in systemic levels of either corticosterone or NGF. Since a submissive behavioral profile is associated with increased risk for injuries due to fighting, it may be that the development of glucocorticoid resistance is an adaptive mechanism that allows the inflammatory component of wound healing to occur in the presence of high levels of corticosterone. Together, these findings demonstrate that the outcomes of social stress may be modified by physiological changes associated with wounding, as well as by behavioral variables such as social status.

Social stress increases the susceptibility to endotoxic shock.

The influence of social disruption stress (SDR) on the susceptibility to endotoxic shock was investigated. SDR was found to increase the mortality of mice when they were challenged with the bacterial endotoxin lipopolysaccharide (LPS). Histological examination of SDR animals after LPS injection revealed widespread disseminated intravascular coagulation in the brain and lung, extensive meningitis in the brain, severe hemorrhage in the lung, necrosis in the liver, and lymphoid hyperplasia in the spleen, indicating inflammatory organ damage. In situ hybridization histochemical analysis showed that the expression of the glucocorticoid receptor mRNA was down-regulated in the brain and spleen of SDR animals while the ratio of expression of AVP/CRH-the two adrenocorticotropic hormone secretagogue, increased. After LPS injection, the expression of pro-inflammatory cytokines, IL-1beta and TNF-alpha, was found significantly higher in the lung, liver, spleen, and brain of the SDR mice as compared with the LPS-injected home cage control animals. Taken together, these results show that SDR stress increases the susceptibility to endotoxic shock and suggest that the development of glucocorticoid resistance and increased production of pro-inflammatory cytokines are the mechanisms for this behavior-induced susceptibility to endotoxic shock.

Multilevel integrative analyses of human behavior: social neuroscience and the complementing nature of social and biological approaches.

Social and biological explanations traditionally have been cast as incompatible, but advances in recent years have revealed a new view synthesized from these 2 very different levels of analysis. The authors review evidence underscoring the complementing nature of social and biological levels of analysis and how the 2 together can foster understanding of the mechanisms underlying complex behavior and the mind. Specifically, they review the utility of considering social influences on biological processes that are often viewed as outside the social domain including genetic constitution, gene expression, disease, and autonomic, neuroendocrine, and immune activity. This research underscores the unity of psychology and the importance of retaining multilevel integrative research that spans molar and molecular levels of analysis. Especially needed in the coming years is more research on the mechanisms linking social and biological events and processes.

Androstenediol-induced restoration of responsiveness to influenza vaccination in mice.

Androstenediol (AED), a metabolite of dehydroepiandrosterone (DHEA) regulates innate and adaptive immune responses. To examine whether AED could effectively reverse the age-associated decline of antiviral immunity, 3-, 10-, and 22-month-old mice were treated with AED-sulfate (AED-S) for 45 days beginning 10 days prior to vaccination. Subsequently, mice were primed and boosted with suboptimal doses of a commercially-available trivalent influenza vaccine. Treatment of 10-month-old animals with AED-S during vaccination increased the titer of circulating antiviral immunoglobulin G to levels comparable with those in 3-month-old mice. Furthermore, AED-S treatment protected 10-month-old animals from intranasal challenge with a lethal dose of influenza virus 21 days after secondary vaccination. Although AED-S treatment of 22-month-old mice did not enhance vaccine responses and failed to protect against lethal challenge, the data from the 10-month-old animals suggest that treatment with AED-S will prevent the early manifestations of immunosenescence.

Steroid hormone regulation of antiviral immunity.

Recent observations in both humans and animals have demonstrated that stress is immunomodulatory and can alter the pathogenesis of microbial infections to the extent that it may be adverse to health. Stress disrupts homeostasis, and the body responds through endocrine and nervous system interactions in an effort to re-establish the health of the host. However, the resulting physiologic changes associated with stress, such as the rise in serum glucocorticoids (GCs), are implicated in suppression of antiviral immunity. Therefore, it would be of significance to counterregulate stress-mediated immunosuppression during viral infection to improve immune responses and limit virus-mediated damage. The data in this study focus upon the antiglucocorticoid influence of a native steroid hormone that has been shown to augment immune function and protect animals against lethal viral infections. Androstenediol (5-androstene-3 beta,17 beta-diol, AED), a metabolite of dehydroepiandrosterone (DHEA), confers protection against lethal infection with influenza A virus. The protective activity appears to counterbalance the function of the regulatory GCs because AED prevents GC-mediated suppression of IL-1, TNF-alpha, and IL-2 secretion. Furthermore, AED inhibits GC-induced transcription of a GC-sensitive reporter gene.

Social disruption, immunity, and susceptibility to viral infection. Role of glucocorticoid insensitivity and NGF.

Glucocorticoid (cort) responses have been shown to suppress inflammatory reactions by inhibiting the trafficking of immune cells. Recently, it was demonstrated that restraint stress (RST) and psychosocial stress (social reorganization; SRO) differentially affected the pathophysiology and survival in the mouse influenza viral infection model. While both stressors activated the HPA axis, only SRO affected survival. In RST, elevated cort diminished recruitment of inflammatory cells following intranasal challenge of C57BL/6 mice with A/PR8 virus. However, infected SRO mice developed hypercellularity in the lungs and were more likely to die from lung consolidation than controls. Since elevated cort failed to be anti-inflammatory in SRO mice, the hypothesis that psychosocial stress induced steroid insensitivity was tested. An in vitro cort suppression test was performed by stimulating splenocytes from SRO and control mice with mitogen in the presence or absence of cort. Proliferation of ConA-stimulated cells was inhibited by cort in a dose-dependent fashion in controls, but splenocytes from SRO mice stimulated with ConA were resistant to cort-induced suppression. Thus, psychosocial stress induced a state of steroid insensitivity. SRO also induced the release of nerve growth factor (NGF) from the salivary glands into circulation; plasma NGF correlated with development of steroid insensitivity. NGF has been reported to negatively regulate the expression of type II glucocorticoid receptors, and thus may be a key factor in the induction of steroid insensitivity.