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The bio-psycho-social model highlights intra-individual and inter-individual interactions, including psychotherapy. The processing of these interactions within a person takes place, among others, in the central autonomic network (CAN). The CAN's autonomic output to the periphery can be indexed by heart rate variability (HRV), representing individual adaptive capacity. Further, the CAN influences the hypothalamus-pituitary-adrenal axis with its product cortisol. The aim consisted in investigating HRV and cortisol as well as their relation to symptom course in response to short-term psychotherapy. A single-arm, uncontrolled, explorative study was conducted at an outpatient psychotherapeutic consultation in the workplace offered to employees with mental or psychosomatic complaints. Questionnaires included symptoms of depression, irritation and functional impairment. Circadian profile of HRV and salivary cortisol concentrations collected pre and post short-term psychotherapeutic intervention (4-12 sessions) were assessed. Multilevel-linear mixed regressions were calculated. Out of 29 participants (mean age 42; 72% female), 24% were on sick leave from work. Cortisol concentrations were neither affected by intervention nor by symptom course. The proportion of individuals showing a vagally mediated HRV in the range of the lowest quartile assessed for age- and sex-matched healthy controls was reduced at follow-up (pre 34%, post 22%; p = .017). Higher vagally mediated HRV at baseline predicted lower symptom burden at follow-up. Thus, the results support the assumption that HRV reflects the capability of an organism to adapt and recover. Patients with reduced HRV might need additional psychotherapeutic sessions to achieve the same symptom improvements than patients with retained HRV.

Chronic psychosocial stress is a risk factor for many mental disorders, including affective disorders, anxiety disorders, and trauma- and stressor-related disorders (i.e., posttraumatic stress disorder, PTSD). As these disorders are associated with an overreactive immune system and chronic low-grade inflammation, immunoregulatory approaches counterbalancing basal and/or stress-induced immune activation should be protective in this context. In support of this hypothesis, we recently demonstrated that repeated subcutaneous (s.c.) preimmunization with a heat-killed preparation of the immunoregulatory bacterium Mycobacterium vaccae (M. vaccae; National Collection of Type Culture (NCTC) 11659) promoted proactive stress coping and protected against stress-induced anxiety and intestinal pathology in a mouse model of chronic psychosocial stress. To induce development of a chronic anxiety-like state, the chronic subordinate colony housing (CSC) paradigm was used. Here we employed the CSC paradigm (start day 1) to confirm the stress-protective effects of repeated s.c. M. vaccae administrations prior to CSC exposure (days -21, -14, and -7) and to extend these findings to the stress-protective role of M. vaccae when administered repeatedly during CSC exposure (days 2, 8 and 15). As readouts we assessed the stress coping behavior on days 1, 8, and 15 and general and/or social anxiety-related behavior on days 19 (elevated plus-maze), 20 (open-field/novel object test), and day 21 (social preference/avoidance test) of CSC exposure. In line with our previous study, M. vaccae administered prior to CSC strongly promoted active stress coping and moderately reduced CSC-induced general and social anxiety. Although M. vaccae administered during CSC did not affect stress coping, this treatment protocol profoundly protected against CSC-induced general, and to a lesser extent social, anxiety. Taken together, these data broaden the framework for developing bioimmunoregulatory approaches, based on the administration of immunoregulatory microorganisms, for the prevention and/or treatment of affective disorders, anxiety disorders, and trauma- and stressor-related psychiatric disorders like PTSD.

Stress-associated somatic and psychiatric disorders are often linked to non-resolving low-grade inflammation, which is promoted at least in part by glucocorticoid (GC) resistance of distinct immune cell subpopulations. While the monocyte/macrophage compartment was in the focus of many clinical and preclinical studies, the role of myeloid-derived suppressor cells (MDSCs) in stress-associated pathologies and GC resistance is less understood. As GC resistance is a clear risk factor for posttraumatic complications in patients on intensive care, the exact interplay of physical and psychosocial traumatization in the development of GC resistance needs to be further clarified. In the current study we employ the chronic subordinate colony housing (CSC) paradigm, a well-characterized mouse model of chronic psychosocial stress, to study the role of myeloid cells, in particular of MDSCs, in innate immune activation and GC resistance following combined psychosocial and physical (e.g., bite wounds) trauma. Our findings support the hypothesis that stress-induced neutrophils, polymorphonuclear (PMN)-MDSCs and monocytes/monocyte-like (MO)-MDSCs get primed and activated locally in the bone marrow as determined by toll-like receptor (TLR)2 upregulation and increased basal and lipopolysaccharide (LPS)-induced in vitro cell viability. These primed and activated myeloid cells emigrate into the peripheral circulation and subsequently, if CSC is accompanied by significant bite wounding, accumulate in the spleen. Here, PMN-MDSCs and monocytes/MO-MDSCs upregulate TLR4 expression, which exclusively in PMN-MDSCs promotes NF-κB hyperactivation upon LPS-stimulation, thereby exceeding the anti-inflammatory capacities of GCs and resulting in GC resistance.

An increasing body of evidence indicates that immunodysregulation and subsequent chronic low-grade inflammation can promote the development of stress-related somatic and psychiatric pathologies, including inflammatory bowel disease (IBD) and posttraumatic stress disorder (PTSD). Thus, immunoregulatory approaches counterbalancing basal and/or stress-induced immune activation should have stress-protective potential. In support of this hypothesis, we recently demonstrated that repeated s.c. preimmunization with a heat-killed preparation of the immunoregulatory bacterium Mycobacterium vaccae ( M. vaccae ; National Collection of Type Culture (NCTC) 11659), protects mice against stress-induced general anxiety, spontaneous colitis, and aggravation of dextran sulfate sodium (DSS)-induced colitis in the chronic subordinate colony housing (CSC) paradigm, a validated model for PTSD in male mice. In the current study, we repeatedly administered M. vaccae via the non-invasive intranasal (i.n.; 0.1 mg/mouse/administration) route, prior to or during CSC exposure or single housed control (SHC) conditions, and assessed the effects on general and social anxiety, and on parameters related to the severity of DSS-induced colitis. While administration of M. vaccae prior to the onset of CSC exposure only had minor stress-protective effects, administration of M. vaccae during CSC completely prevented CSC-induced aggravation of DSS colitis. As CSC in the current experimental setting did not reliably increase general anxiety-related behavior, potential stress-protective effects of M.vaccae are difficult to interpret. Taken together, these data broaden the framework for developing bioimmunoregulatory approaches, based on the administration of microorganisms with anti-inflammatory and immunoregulatory properties, for the prevention of stress-related disorders.

Chronic subordinate colony housing (CSC), an established mouse model for chronic psychosocial stress, promotes a microbial signature of gut inflammation, characterized by expansion of Proteobacteria, specifically Helicobacter spp., in association with colitis development. However, whether the presence of Helicobacter spp. during CSC is critically required for colitis development is unknown. Notably, during previous CSC studies performed at Regensburg University (University 1), male specific-pathogen-free (SPF) CSC mice lived in continuous subordination to a physically present and Helicobacter spp.-positive resident. Therefore, it is likely that CSC mice were colonized, during the CSC procedure, with Helicobacter spp. originating from the dominant resident. In the present study we show that employing SPF CSC mice and Helicobacter spp.-free SPF residents at Ulm University (University 2), results in physiological responses that are typical of chronic psychosocial stress, including increased adrenal and decreased thymus weights, decreased adrenal in vitro adrenocorticotropic hormone (ACTH) responsiveness, and increased anxiety-related behavior. However, in contrast to previous studies that used Helicobacter spp.-positive resident mice, use of Helicobacter spp.-negative resident mice failed to induce spontaneous colitis in SPF CSC mice. Consistent with the hypothesis that the latter is due to a lack of Helicobacter spp. transmission from dominant residents to subordinate mice during the CSC procedure, colonization of SPF residents with Helicobacter typhlonius at University 2, prior to the start of the CSC model, rescued the colitis-inducing potential of CSC exposure. Furthermore, using SPF CSC mice and H. typhlonius-free SPF residents at University 1 prevented CSC-induced colitis. In summary, our data support the hypothesis that the presence or absence of exposure to certain pathobionts contributes to individual variability in susceptibility to stress-/trauma-associated pathologies and to reproducibility of stress-related outcomes between laboratories.

While animal housing conditions are highly controlled and standardized between different laboratories, there are still many subtle differences that unavoidably influence the host organisms and, consequently, interlaboratory reproducibility. Here, we investigated the physiological and immunological consequences between two light/dark cycle (LDC) lengths (14-h/10-h vs. 12-h/12-h LDC) and two commonly used forms of drinking water (acidified drinking water (AW) versus normal tap water (NW)) in single-housed (SH) mice. Our results indicate that SH mice bred under a 12-h/12-h LDC and NW at the supplier's facility showed increased basal morning plasma corticosterone (CORT) levels even 4 weeks after arrival at our animal facility employing a 14-h/10-h LDC and AW. This effect was even more pronounced two weeks after arrival and had abated after 8 weeks. In agreement, increased plasma adrenocorticotropic hormone (ACTH), adrenal in vitro ACTH sensitivity, as well as relative and absolute adrenal weight normalized during this 8-week exposure to the novel and unfamiliar 14-h/10-h LDC and AW. Employment of a 12-h/12-h LDC in our facility completely abrogated the CORT-elevating effects of the 14-h/10-h LDC, despite these animals drinking AW. When both the water and light conditions were matched to those at the supplier's facility, we observed a further reduction in adrenal weight, increased thymus weight, and decreased pro-inflammatory cytokine secretion of isolated and anti-CD3/28-stimulated mesenteric lymph node cells. In summary, our results indicate that prolonged alteration of both the light phase and drinking water represent severe and long-lasting stressors for laboratory rodents. These findings are of general interest for all scientists obtaining their experimental animals from conventional suppliers.

Background and aim: Chronic subordinate colony housing (CSC, 19 days), an established and preclinically-validated mouse model for posttraumatic stress disorder (PTSD), causes evening hypocorticism and a reduced adrenal in vitro ACTH (adrenocorticotropic hormone) sensitivity despite pronounced adrenal hyperplasia. However, until now it remains unclear at what time point during CSC exposure evening hypocorticism and adrenal in vitro ACTH insensitivity develop and whether the repeated change of dominant aggressor mice plays an important role in this context. It is, therefore, the aim of the current study, to explore the detailed time course of these stress-induced adrenal changes. Methods: Adrenal weight, plasma corticosterone (CORT) and ACTH were assessed in the morning of days 8 (right before exposure to the 2nd aggressor), 9 (24 h after exposure to the 2nd aggressor), 15 (right before exposure to the 3rd aggressor), 16 (24 h after exposure to the 3rd aggressor) and 20 or in the evening of days 8 (10 h after exposure to the 2nd aggressor), 9 (34 h after exposure to the 2nd aggressor), 15 (10 h after exposure to the 3rd aggressor), 16 (34 h after exposure to the 3rd aggressor) and 20 of CSC exposure. Moreover, we in vitro cultured adrenal explants of all mice euthanized in the morning of days 8, 9, 15, 16 and 20 either in the presence or absence of ACTH to subsequently assess CORT concentration in the supernatants. Results: Our results indicate that while adrenal mass was increased at all time points assessed, plasma morning CORT only transiently increased in response to the 2nd (on day 8) but not 3rd (on day 15) dominant aggressor mouse. Moreover, although mild signs of adrenal in vitro ACTH insensitivity developed already after one week of CSC exposure, moderate and severe adrenal in vitro ACTH insensitivity required two and three weeks of chronic subordination, respectively. Conclusion: Together with unaffected plasma ACTH levels at all time points assessed, our data suggest that stress-induced adrenal in vitro ACTH insensitivity develops gradually during times of chronic subordination while subordination to different aggressor mice aggravates its severity. Moreover, a mild form of adrenal ACTH insensitivity seems to allow prevention of morning hypercorticism on day 8 of CSC, despite functional adrenal mass being increased, while a moderate and severe form of adrenal ACTH insensitivity in CSC mice seems to promote HPA axis adaptation to repeated homotypic stressor exposure (i.e. dominant aggressor mice) and basal evening hypocorticism in CSC mice, respectively. Our results might, therefore, be the basis for future clinical studies assessing CORT supplementation as novel treatment regimen for somatic and affective pathologies linked to chronic and/or traumatic stress.

Although chronic stress is an acknowledged risk factor for the development of somatic and affective disorders, the cellular and molecular mechanisms underlying stress-induced pathologies are not fully understood. Interestingly, rodent studies involving immune cell transfer suggest that CD4+ T cells might be at least in part involved in reactivation of a chemically-induced colitis by stress. However, until now evidence is lacking that these immune cell types are indeed involved in the development of a "stressed phenotype". The aim of the present study was, therefore, to assess the effects of adoptively transferring total mesenteric lymph node cells (mesLNCs) and CD4+ mesLNCs isolated from chronically-stressed mice into healthy recipient mice on various physiological, immunological and behavioral parameters. To induce chronic psychosocial stress in donor mice we employed the chronic subordinate colony housing (CSC) paradigm. Our data indicate that transfer of total or CD4+ mesLNCs from CSC mice, compared with respective cells from single-housed control (SHC) mice, promoted splenomegaly and interferon (IFN)-γ secretion from in vitro anti-CD3-stimulated mesLNCs in naive recipient mice. This effect was independent of recipient mice additionally being administered with dextran sulfate sodium (DSS) or not. Transfer of CD4+ mesLNCs additionally increased adrenal weight and secretion of IL-6 from in vitro anti-CD3 stimulated mesLNCs in recipients administered with DSS. Importantly, transfer of neither cell type from CSC vs. SHC donor mice affected anxiety-related behavior of recipient mice in the light-dark box. Taken together, our data demonstrate that typical physiological and immunological, but not behavioral, effects of chronic stress can be induced in naive recipient mice by adoptively transferring mesLNCs, in particular CD4+ mesLNCs, from chronically stressed donor mice.

Cardiovascular disorders (CVD) and posttraumatic stress disorder (PTSD) are highly comorbid, but the underlying mechanisms are not fully understood. Chronic psychosocial stress was induced in male mice by chronic subordinate colony housing (CSC), a pre-clinically validated mouse model for PTSD. Cardiac structure and function were assessed on day 20 of the CSC paradigm. Following CSC, mice were kept in different sensory contact modalities to the last aggressor for 30 days, and development of cardiac function and behavioral aspects were determined. Here we show that psychosocial trauma affects heart structure by disturbing cell-to-cell integrity of cardiomyocytes, causes tachycardia, disturbance of diurnal heart rate rhythmicity and behavioral deficits in a mouse model for PTSD. Structural and functional alterations were also found in cardiomyocytes upon in vitro treatment with pro-inflammatory cytokines typically increased after psychosocial trauma. Interestingly, sensory contact to the aggressor subsequent to psychosocial trauma prohibits functional and structural heart recovery, while isolation was beneficial for cardiac but detrimental for mental health. These findings contribute to our understanding of potential mechanisms underlying the high comorbidity of CVD and PTSD.

Increased basal glucocorticoid secretion and a reduced glucocorticoid response during acute stress, despite only minor changes in the secretion of the major secretagogue adrenocorticotropic hormone (ACTH), have been documented in the peripartum period in several species. We recently showed that the adrenal gland, the site of glucocorticoid synthesis, undergoes substantial postpartum-associated plasticity in the rat at mid-lactation. Here, we asked the question whether adrenal changes already take place around parturition in the rat and in another species, namely the mouse. After demonstrating that several components of the adrenal machinery mediating cholesterol supply for steroidogenesis, including protein levels of hormone-sensitive lipase, low-density lipoprotein receptor (LDLR) and scavenger receptor class-B type-1 (SRB1), are upregulated, while hydroxymethylglutaryl coenzyme A reductase (HMGCR) is downregulated in the lactating rat one day after delivery, as previously observed at mid-lactation, we demonstrated profound changes in the mouse. In detail, protein expression of LDLR, SRB1, HMGCR and adrenal lipid store density were increased in the mouse adrenal one day after parturition as tested via western blot analysis and oil-red lipid staining, respectively. Moreover, using in vitro culture techniques, we observed that isolated adrenal explants from lactating mice secreted higher levels of corticosterone under basal conditions, but showed impaired responsiveness to ACTH, mimicking the in vivo scenario. These results suggest that mechanisms of adaptation in the maternal adrenal after delivery, namely increased cholesterol availability and decreased ACTH sensitivity, are crucial for the basal increase in circulating glucocorticoids and maternal stress hyporesponsiveness that are typical of this period.

Mice exposed to chronic subordinate colony housing (CSC, 19 days) show an exaggerated adrenal corticosterone response to an acute heterotypic stressor (elevated platform (EPF), 5 min) despite no difference from EPF-exposed single-housed control (SHC) mice in corticotropin (ACTH) secretion. In the present study, we asked the question whether this CSC-induced increase in adrenal capability to produce and secrete corticosterone is paralleled by an enhanced adrenal availability and/or mobilization capacity of the corticosterone precursor molecule cholesterol. Employing oil-red staining and western blot analysis we revealed comparable relative density of cortical lipid droplets and relative protein expression of hormone-sensitive lipase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and low-density lipoprotein receptor (LDL-R) between CSC and SHC mice. However, relative protein expression of the scavenger receptor class B type 1 (SR-BI) was increased following CSC exposure. Moreover, analysis of plasma high-density lipoprotein-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) revealed increased LDL-C levels in CSC mice. Together with the pronounced increase in adrenal weight, evidently mediated by hyperplasia of adrenocortical cells, these data strongly indicate an enhanced adrenal availability of and capacity to mobilize cholesterol in chronic psychosocially-stressed mice, contributing to their increased in vivo corticosterone response during acute heterotypic stressor exposure.

There is considerable individual variability in vulnerability for developing posttraumatic stress disorder (PTSD); evidence suggests that this variability is related in part to genetic and environmental factors, including adverse early life experience. Interestingly, recent studies indicate that induction of chronic low-grade inflammation may be a common mechanism underlying gene and environment interactions that increase the risk for development of PTSD symptoms, and, therefore, may be a target for novel interventions for prevention or treatment of PTSD. Development of murine models with face, construct, and predictive validity would provide opportunities to investigate in detail complex genetic, environmental, endocrine, and immunologic factors that determine vulnerability to PTSD-like syndromes, and furthermore may provide mechanistic insight leading to development of novel interventions for both prevention and treatment of PTSD symptoms. Here we describe the potential use of the chronic subordinate colony housing (CSC) paradigm in mice as an adequate animal model for development of a PTSD-like syndrome and describe recent studies that suggest novel interventions for the prevention and treatment of PTSD.

Etiology and pharmacotherapy of stress-related psychiatric conditions and somatoform disorders are areas of high unmet medical need. Stressors holding chronic plus psychosocial components thereby bear the highest health risk. Although the metabotropic glutamate receptor subtype 5 (mGlu5) is well studied in the context of acute stress-induced behaviors and physiology, virtually nothing is known about its potential involvement in chronic psychosocial stress. Using the mGlu5 negative allosteric modulator CTEP (2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4yl]ethynyl]pyridine), a close analogue of the clinically active drug basimglurant – but optimized for rodent studies, as well as mGlu5-deficient mice in combination with a mouse model of male subordination (termed CSC, chronic subordinate colony housing), we demonstrate that mGlu5 mediates multiple physiological, immunological, and behavioral consequences of chronic psychosocial stressor exposure. For instance, CTEP dose-dependently relieved hypothalamo-pituitary-adrenal axis dysfunctions, colonic inflammation as well as the CSC-induced increase in innate anxiety; genetic ablation of mGlu5 in mice largely reproduced the stress-protective effects of CTEP and additionally ameliorated CSC-induced physiological anxiety. Interestingly, CSC also induced an upregulation of mGlu5 in the hippocampus, a stress-regulating brain area. Taken together, our findings provide evidence that mGlu5 is an important mediator for a wide range of chronic psychosocial stress-induced alterations and a potentially valuable drug target for the treatment of chronic stress-related pathologies in man.

A better neurobiological understanding of high and abnormal aggression based on adequate animal models is essential for novel therapy and prevention. Selective breeding of rats for extremes in anxiety-related behavior resulted in two behavioral phenotypes with high and abnormal forms of aggression. Rats bred for low anxiety-related behavior (LAB) consistently show highest levels of aggression and little social investigation in the resident-intruder (RI) test, compared with non-selected low-aggressive (NAB) rats. High anxiety-related (HAB) rats also show higher levels of aggression than NAB rats, but to a lesser extent than LAB rats. Accordingly, extremes in inborn anxiety in both directions are linked to an increased aggression level. Further, both LAB and HAB, but not NAB males, display abnormal aggression (attacks towards vulnerable body parts, females or narcotized males), which is particularly prominent in LABs. Also, only in LAB rats, the nucleus accumbens (NAc) was found to be strongly activated in response to the RI test as reflected by increased c-fos and zif268 mRNA expression, and higher local dopamine release compared with NAB males, without differences in local dopamine receptor binding. Consequently, local pharmacological manipulation by infusion of an anesthetic (lidocaine, 20 μg/μl) or a dopamine D2 (haloperidol, 10 ng/μl), but not D1 (SCH-23390 10 ng/μl), receptor antagonist significantly reduced high aggression in LAB rats. Thus, LAB rats are an adequate model to study high and abnormal aggression. In LAB males, this is likely to be linked to hyper-activation of the reward system, as found in psychopathic patients. Specifically, activation of the accumbal dopamine system is likely to underlie the high aggression observed in LAB rats.

Summary Although chronic psychosocial stress is often accompanied by changes in basal hypothalamo-pituitary-adrenal (HPA) axis activity, it is vital for a chronically-stressed organism to mount adequate glucocorticoid (GC) responses when exposed to acute challenges. The main aim of the present study was to test whether this is true or not for the chronic subordinate colony housing (CSC, 19 days) paradigm, an established and clinically relevant mouse model of chronic psychosocial stress. As shown previously, CSC mice are characterized by unaffected morning and decreased evening plasma corticosterone (CORT) levels despite enlarged adrenals, suggesting a maladaptive breakdown of adrenal functioning. Plasma CORT levels, determined by repeated blood sampling via jugular vein catheters, as well as relative right adrenal CORT content were increased in CSC compared with single-housed control (SHC) mice in response to acute elevated platform (EPF, 5 min) exposure. However, in vitro stimulation of adrenal explants with physiological and pharmacological doses of ACTH revealed an attenuated responsiveness of both the left and right adrenal glands following CSC, despite mRNA and/or protein expression of melanocortin 2 receptor (Mc2r), Mc2r accessory protein (MRAP), and key enzymes of steroidogenesis were not down-regulated. Taken together, we show that chronic psychosocial stressor exposure impairs in vitro ACTH responsiveness of both the left and right adrenal glands, whereas it increases adrenal responsiveness to an acute heterotypic stressor in vivo . This suggests that an additional factor present during acute stressor exposure in vivo rescues left and right adrenal ACTH sensitivity, or itself acts as CORT secretagogue in chronically stressed CSC mice.

Chronic stress, in particular chronic psychosocial stress, is a risk factor in the aetiology of various psychopathologies including anxiety- and depression-related disorders. Therefore, recent studies have focussed on the development of social-stress paradigms, which are believed to be more relevant to the human situation than non-social-stress paradigms. The majority of these paradigms have been reported to increase both anxiety- and depression-related behaviour in rats or mice. However, in order to dissect the mechanisms underlying anxiety or depression, animal models are needed, which specifically induce one, or the other, phenotype. Here, we study both short- (1d after stressor termination) and long-term (4d or 7d after stressor termination) behavioural and physiological consequences of two well-validated chronic psychosocial stress models: social-defeat/overcrowding (SD/OC) and chronic subordinate colony housing (CSC). We demonstrate that SD/OC and CSC result in different physiological alterations: SD/OC more strongly affecting body-weight development, whereas CSC more strongly affects adrenal and pituitary morphology. Both stressors were shown to flatten circadian locomotor activity immediately after stress termination, which normalized 7d later in SD/OC group but reversed to hyperactivity during the dark phase in the CSC group. Importantly, neither stress paradigm resulted in an increase in depression-related behaviour as assessed using the forced swim test, tail suspension test and saccharin preference test at any time-point. However, both stress paradigms lead to an anxiogenic phenotype; albeit with different temporal profiles and not towards a novel con-specific (social anxiety). CSC exposure elevates anxiety-related behaviour immediately after stressor termination, which lasts for at least 1 wk. In contrast, the anxiogenic phenotype only develops 1 wk after SD/OC termination. In conclusion, both models are unique for uncovering the molecular underpinnings of anxiety-related behaviour without conflicting depression-based alterations.

Patients with inflammatory bowel diseases (IBDs) have a higher risk of developing colorectal cancer (CRC) than the general population. Furthermore, chronic psychosocial stress increases the likelihood of developing IBD and multiple types of malignant neoplasms, including CRC. Here, for the first time, we investigate the effects of chronic psychosocial stress in male mice on an artificially induced CRC, by employing the chronic subordinate colony (CSC) housing paradigm in combination with the reliable azoxymethane (AOM)/dextran sodium sulfate (DSS) CRC model. Colonoscopy revealed that CSC mice showed accelerated macroscopic suspect lesions. In addition, more CSC mice developed low-grade dysplasia (LGD) and/or high-grade dysplasia (HGD) in the colonic tissue compared to the single-housed control mice (SHC). CSC mice showed an increased number of Ki67+ and a decreased number of terminal deoxynucleotidyl transferase dUTP nick end labeling epithelial cells in colonic tissue. Colonic liver receptor homolog-1 (LRH-1), cyclooxygenase II (COXII), tumor necrosis factor, forkhead box P3 (FoxP3) mRNA as well as colonic ß-catenin, COXII, and LRH-1 protein expression were also increased in CSC compared with SHC mice. Although the number of CD4+ Th cells was increased, a tendency toward a decreased colonic interferon-γ (IFN-γ) mRNA expression was observed. Furthermore, despite an increased percentage of CD3+ cells and CD3+/FoxP3+ double-positive cells within mesenteric lymph node cells of CSC mice, IFN-γ secretion from these cells was unaffected. Altogether, our results suggest that chronic psychosocial stress increases the risk for AOM/DSS-induced and, thus, inflammation-related CRC. Finally, assessment of additional time points may test whether the shift from tumor-protective Th1 cell to regulatory T-cell immunity represents a consequence of increased carcinogenesis or a causal factor involved in its development.

Chronic psychosocial stress is a risk factor for many affective and somatic disorders, including inflammatory bowel diseases. In support chronic subordinate colony housing (CSC, 19 days), an established mouse model of chronic psychosocial stress, causes the development of spontaneous colitis. However, the mechanisms underlying the development of such stress-induced colitis are poorly understood. Assessing several functional levels of the colon during the initial stress phase, we show a pronounced adrenal hormone-mediated local immune suppression, paralleled by impaired intestinal barrier functions, resulting in enhanced bacterial load in stool and colonic tissue. Moreover, prolonged treatment with broad-spectrum antibiotics revealed the causal role of these early maladaptations in the development of stress-induced colitis. Together, we demonstrate that translocation of commensal bacteria is crucial in the initiation of stress-induced colonic inflammation. However, aggravation by the immune-modulatory effects of fluctuating levels of adrenal hormones is required to develop this into a full-blown colitis.

Chronic subordinate colony (CSC) housing has been recently validated as a murine model of chronic psychosocial stress which induces alterations of stress-related parameters including decreased body-weight gain and an increased level of anxiety in comparison with single housed control (SHC) mice. By using immunohistochemical immediate early gene (IEG) mapping we investigated whether CSC housing causes alterations in neuronal activation patterns in limbic areas including the amygdala, hippocampus, septum and the periaqueductal gray (PAG) and hypothalamic paraventricular nucleus (PVN). While CSC housing increased basal Zif-268 expression in the nucleus accumbens shell compared to SHC, IEG responses to subsequent open arm (OA) exposure were attenuated in the ventral and intermediate sub-regions of the lateral septum, parvocellular PVN and the dorsal CA3 region of the hippocampus of CSC compared with SHC mice. In contrast, a potentiated c-Fos response in CSC mice was observed in the dorsomedial PAG after OA exposure. Confirming previous findings obtained on the elevated plus-maze, an enhanced anxiety-related behavior in CSC compared with SHC mice was also observed during OA exposure. In order to investigate the appropriate control conditions for CSC housing, group housed control (GHC) mice were additionally included in the behavioral testing. Interestingly, GHC as well as CSC mice showed significantly less risk assessment/exploratory behavior during OA exposure compared with SHC mice indicating that group housing itself is stressful for mice and not an adequate control for the CSC paradigm. Overall, CSC housing is an ethologically relevant chronic psychosocial stressor which results in an elevated sensitivity to a subsequent novel, aversive challenge. However, the CSC-induced increase in anxiety-related behavior was accompanied by differences in neuronal activation, compared with SHC, in defined sub-regions of brain areas known to be involved in the processing of emotionality and stress responses.

A commonly used method for obtaining blood samples from mice is decapitation. However, there is an obvious need for repeated blood sampling in mice under stress-free conditions. Here, we describe a simple technique to repeatedly collect blood samples from conscious, freely moving mice through a chronically implanted jugular vein catheter. Furthermore, we compare plasma corticosterone (CORT) concentrations in samples obtained through the catheter 1 day after surgery with samples taken from trunk blood obtained under basal or acute stress conditions. CORT concentrations in repeated 100-μl venous blood samples were found to be similar to trunk blood samples both under basal conditions and after stressor exposure collected at identical time points (at 5, 15, and 60 min). Using both techniques, we demonstrate a progressive increase in CORT levels until 15 min after termination of stressor exposure and a decrease towards baseline values 60 min later. Anxiety-related behavior, as assessed on the elevated plus maze 3-4 days after surgery, did not differ between catheterized and non-catheterized mice. Our results provide evidence for application of jugular vein catheterization as a technique for repeated blood sampling in conscious laboratory mice. Use of this technique will greatly reduce the number of animals required for experiments involving endocrine endpoints.

Chronic psychosocial stress has been repeatedly shown in humans to be a risk factor for the development of several affective and somatic disorders, including inflammatory bowel diseases (IBD). There is also a large body of evidence from rodent studies indicating a link between stress and gastrointestinal dysfunction, resembling IBD in humans. Despite this knowledge, the detailed underlying neuroendocrine mechanisms are not sufficiently understood. This is due, in part, to a lack of appropriate animal models, as most commonly used rodent stress paradigms do not adequately resemble the human situation and/or do not cause the development of spontaneous colitis. Therefore, our knowledge regarding the link between stress and IBD is largely based on rodent models with low face and predictive validity, investigating the effects of unnatural stressors on chemically induced colitis. These studies have consistently reported that hypothalamo-pituitary-adrenal (HPA) axis activation during stressor exposure has an ameliorating effect on the severity of a chemically induced colitis. However, to show the biological importance of this finding, it needs to be replicated in animal models employing more clinically relevant stressors, themselves triggering the development of spontaneous colitis. Important in view of this, recent studies employing chronic/repeated psychosocial stressors were able to demonstrate that such stressors indeed cause the development of spontaneous colitis and, thus, represent promising tools to uncover the mechanisms underlying stress-induced development of IBD. Interestingly, in these models the development of spontaneous colitis was paralleled by decreased anti-inflammatory glucocorticoid (GC) signaling, whereas adrenalectomy (ADX) prior to stressor exposure prevented its development. These findings suggest a more complex role of the HPA axis in the development of spontaneous colitis. In the present review I summarize the available human and rodent data in order to provide a comprehensive understanding of the biphasic role of the HPA axis and/or the GC signaling during stressor exposure in terms of spontaneous colitis development.

Recent studies indicate that chronic psychosocial stress favors the development of generalized immune dysfunction. During stressor exposure neuroendocrine factors affect numbers and functionality of leukocytes. However, the exact mechanisms leading to systemic changes in immune functions during stress are still not clear. During chronic subordinate colony housing, a model of chronic psychosocial stress, mice developed spontaneous colonic inflammation. Decreased glucocorticoid signaling, induced by a combination of adrenal insufficiency and glucocorticoid resistance, was thought to prevent tempering of local immune cells, and to promote tissue inflammation. In this study we investigated changes in the systemic immune status after chronic subordinate colony housing and analyzed potential mechanisms underlying those alterations. Analysis of T helper cell subsets in peripheral lymph nodes revealed a reduction of regulatory T cells, accompanied by increased T cell effector functions. Generalized activation of T cells was shown by elevated cytokine production upon stimulation. In addition, we observed no apparent shift towards T helper type 2 responses. It is likely, that the previously reported hypocorticism in this stress model led to a steady production of inflammatory Th1, Th2, and Th17 cytokines and obstructed the shift towards an anti-inflammatory response. In conclusion, we established chronic subordinate colony housing as a model to investigate the outcome of stress on the systemic immune status. We also provide evidence that distinct T helper cell subtypes react differentially to the suppressive effect of glucocorticoids.

The correlation between chronic stress and aggravation of inflammatory diseases has long been implied. A variety of studies in men and mice have proven the increase in myeloid cell blood counts as a hallmark of stressor exposure. Redistribution of myeloid cells from primary and secondary lymphoid organs into the blood is thought to be the major reason for that phenomenon. Recently, we established a model of chronic subordinate colony housing (CSC) in male mice. CSC induced an aggravation of DSS-induced colitis. Gut inflammation was accompanied with an increased translocation of commensal bacteria from the gut lumen into the surrounding tissue. We now analysed changes in the composition and function of myeloid cell subtypes in spleen and gut. CSC stress induced an increase in myeloid cells in blood, spleen and gut. A detailed analysis revealed that CD11b + cells in the spleen consisted mainly of Ly6G + granulocytic cells. Furthermore, myeloid cells from the spleen reacted with an increased cytokine production to in vitro restimulation with LPS indicating an inflammatory phenotype of those cells. CSC also induced up-regulation of CXCL1 and CXCL2 in the spleen and in the gut. Our study showed a stress-induced accumulation of distinct myeloid cells in secondary lymphoid organs and peripheral tissue that seemed to be mediated by specific chemokines induced during exposure to CSC. Due to their inflammatory phenotype these cells might contribute to the aggravation of local gut inflammation seen in the DSS-induced colitis.

Chronic subordinate colony housing (CSC) is an adequate mouse model of chronic psychosocial stress, besides other behavioural and physiological consequences inducing development of spontaneous colitis. The latter is characterized by an increased histological damage score and activation of T cells in the draining mesenteric lymph nodes (mesLN). Here, we isolated total mesLN cells (mesLNC) from CSC and single housed control (SHC) mice, separated CD4 + mesLNC, quantified their anti-CD3-stimulated interferon-γ (IFN-γ) secretion, and adoptively transferred 3 × 10 6 of these cells into naive syngenic C57BL/6 male recipient mice. One week later, recipient mice were killed and selected physiological and immunological parameters assessed. While the number of mesLNC was more than threefold increased following CSC, the percentage of CD4 + cells was strongly decreased in CSC (14.5 %) compared with SHC (27.3 %) donor mice. However, CD4 + mesLNC of CSC compared with SHC donor mice produced significantly more IFN-γ. Furthermore, CSC recipient mice showed increased absolute spleen and adrenal weights, plasma corticosterone concentrations, numbers of total mesLNC, and IFN-γ secretion from these cells during anti-CD3 stimulation. Currently, we are assessing the cellular composition of mesLNC in CSC and SHC donor mice by flow cytometry and the colitogenic potential of adoptively transferred CSC and SHC CD4 + mesLNC in RAG-1-deficient mice, respectively. Together, our results indicate that transfer of a single cell population from chronic psychosocially-stressed mice induces an overall pro-inflammatory milieu in naive recipient mice, probably contributing to an overall risk for developing inflammatory disorders, i.e. colitis.

Inflammatory bowel disease (IBD) is a chronic disorder with widespread ethiology; genetic predisposition, nutrition, and the immune state are factors influencing the outcome of the disease. Furthermore, chronic stress has negatively been linked to IBD. Chronic subordinate colony housing (CSC) has recently been established as a model to study the impact of stress on colonic inflammation. So far, this model correlates stressor exposure with pathophysiological changes in the gut. However, reasons for those changes are still poorly understood. As myeloid cells play a critical role in IBD we focused on the analysis of stress-induced myeloid cells in CSC. Early during stressor exposure an increase in the myeloid cell growth factor G-CSF was observed. Consequently, a steady increase of myeloid cells was detected in secondary lymphoid organs and the colon. The cells were further characterized by the expression of CD11b and Gr1 identifying them as inflammatory myeloid cells (IMC). Stress-derived IMC produced high amounts of inflammatory cytokines upon stimulation indicating their pathological role in that particular context. Furthermore, we were able to reproduce the increase in IMC and G-CSF by application of glucocorticoids. In conclusion, our study demonstrates that chronic psychosocial stress induces distinct changes in the myeloid cell compartment that stimulates accumulation of IMC – a cell type critical for the development of colonic inflammation.

Stress has been considered to negatively affect the outcome of cancer. In the physiological stress response neurotransmitters and hormones modulate immune cell function that in turn alters the tumor environment, thus, modulates tumor growth. In particular, myeloid-derived suppressor cells (MDSC) that were initially identified in tumor patients and are characterized by the expression of CD11b, Ly6G and Ly6C seem to play a critical role in the promotion of tumor growth. As the increase in CD11b-positive cells in general is a hallmark of exposure to stress we sought to investigate the impact of chronic psychosocial stress on MDSC development and subsequent tumor growth. We applied chronic subordinate colony housing (CSC), a previously established paradigm for chronic psychosocial stress. We analyzed the phenotype and function of CD11b-positive myeloid cells accumulating in stressed mice as well as those arising in response to an external tumor. After 19 days of CSC myeloid cells were increased in blood and secondary lymphoid organs. Detailed analysis of such cells revealed their MDSC-like phenotype. Consistently, we observed enhanced tumor-growth in stressed mice. Previous exposure to CSC enhanced suppressive activity of MDSC in tumor-bearing mice. In conclusion, our data indicate that exposure to chronic psychosocial stress induces the development of MDSC, that gain functional activity in tumor-bearing mice, thus, promoting tumor growth.

Chronic psychosocial stress is an accepted risk factor for various affective and somatic disorders. In an established murine chronic stress model, chronic subordinate colony housing (CSC) results in the alteration of physiological, behavioural, neuroendocrine and immunological parameters (1). Accompanied by thymus atrophy and adrenal hypertrophy as indicators of chronic stress, CSC mice show impaired glucocorticoid signalling and increased pro-inflammatory cytokine secretion from mesenteric lymph node cells (mesLNC), all contributing to CSC-induced spontaneous colitis, aggravation of chemically induced colitis and the enhanced risk for developing colorectal cancer (2). Further, CSC mice show a long-lasting increase in general anxiety, whereas, in contrast, depression-related behaviours remain unchanged (3). First experiments indicate that chronic icv administration of oxytocin, a neuropeptide with anxiolytic and anti-stress effects (4), via osmotic minipumps during CSC exposure dose-dependently affects some of the CSC-induced consequences. For example, at low dose (1 ng/h), chronic oxytocin prevented thymus atrophy, adrenal hypertrophy and the interferon gamma hyper secretion from mesLNC. In contrast, chronic high doses of oxytocin (10 ng/h) increased anxiety-related behaviour in unstressed mice and reduced oxytocin receptor binding in limbic brain areas. Thus, administration of oxytocin during ongoing chronic stress seems to partly prevent stress-induced mal-adaptations. 1 Reber S et al. Endocrinology 2007, 148:670–82. 2 Peters S et al. Stress 2012, 15:403–15 3 Slattery DA et al. Psychoneuroendocrinology 2012, 37:702–14 4 Neumann ID and Landgraf R. TINS 2012, 35:649–659

Animal models of chronic social stress are known to contribute to a great extent to the research of stress-related disorders in humans. Our previous data revealed a generalized activation of T cells and an altered reaction of T cell subtypes not showing a shift towards Th2 responses after 19 days of chronic subordinate colony housing (CSC), a chronic psycho-social stress model in male mice. In this study, we evaluated the time-dependent effects of CSC on the T cell activation status. Our results demonstrated changes in T cell composition concerning CD3, CD8, CD4 as well as Treg, Th1, and Th17 cells. In addition, the activation status of T cells differed during the time of CSC. In conclusion, CSC stress induced distinct changes in the T cell compartment depending on the duration of stress exposure.