Stefan O. Reber

Immunization with a heat-killed preparation of the environmental bacterium Mycobacterium vaccae promotes stress resilience in mice

Significance The hygiene, or “old friends,” hypothesis proposes that lack of exposure to immunoregulatory microorganisms in modern urban societies is resulting in an epidemic of inflammatory disease, as well as psychiatric disorders in which chronic, low-level inflammation is a risk factor. An important determinant of immunoregulation is the microbial community occupying the host organism, collectively referred to as the microbiota. Here we show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Treatment of mice with a heat-killed preparation of an immunoregulatory environmental microorganism, Mycobacterium vaccae, prevents stress-induced pathology. These data support a strategy of “reintroducing” humans to their old friends to promote optimal health and wellness. The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Repeated immunization with a heat-killed preparation of Mycobacterium vaccae, an immunoregulatory environmental microorganism, reduced subordinate, flight, and avoiding behavioral responses to a dominant aggressor in a murine model of chronic psychosocial stress when tested 1–2 wk following the final immunization. Furthermore, immunization with M. vaccae prevented stress-induced spontaneous colitis and, in stressed mice, induced anxiolytic or fear-reducing effects as measured on the elevated plus-maze, despite stress-induced gut microbiota changes characteristic of gut infection and colitis. Immunization with M. vaccae also prevented stress-induced aggravation of colitis in a model of inflammatory bowel disease. Depletion of regulatory T cells negated protective effects of immunization with M. vaccae on stress-induced colitis and anxiety-like or fear behaviors. These data provide a framework for developing microbiome- and immunoregulation-based strategies for prevention of stress-related pathologies.

The Microbiota, Immunoregulation, and Mental Health: Implications for Public Health

The hygiene or “Old Friends” hypothesis proposes that the epidemic of inflammatory disease in modern urban societies stems at least in part from reduced exposure to microbes that normally prime mammalian immunoregulatory circuits and suppress inappropriate inflammation. Such diseases include but are not limited to allergies and asthma; we and others have proposed that the markedly reduced exposure to these Old Friends in modern urban societies may also increase vulnerability to neurodevelopmental disorders and stress-related psychiatric disorders, such as anxiety and affective disorders, where data are emerging in support of inflammation as a risk factor. Here, we review recent advances in our understanding of the potential for Old Friends, including environmental microbial inputs, to modify risk for inflammatory disease, with a focus on neurodevelopmental and psychiatric conditions. We highlight potential mechanisms, involving bacterially derived metabolites, bacterial antigens, and helminthic antigens, through which these inputs promote immunoregulation. Though findings are encouraging, significant human subjects’ research is required to evaluate the potential impact of Old Friends, including environmental microbial inputs, on biological signatures and clinically meaningful mental health prevention and intervention outcomes.

Chronic Subordinate Colony Housing Paradigm: A Mouse Model to Characterize the Consequences of Insufficient Glucocorticoid Signaling

Chronic, in particular chronic psychosocial, stress is a burden of modern societies and known to be a risk factor for numerous somatic and affective disorders (in detail referenced below). However, based on the limited existence of appropriate, and clinically relevant, animal models for studying the effects of chronic stress, the detailed behavioral, physiological, neuronal, and immunological mechanisms linking stress and such disorders are insufficiently understood. To date, most chronic stress studies in animals employ intermittent exposure to the same (homotypic) or to different (heterotypic) stressors of varying duration and intensity. Such models are only of limited value, since they do not adequately reflect the chronic and continuous situation that humans typically experience. Furthermore, application of different physical or psychological stimuli renders comparisons to the mainly psychosocial stressors faced by humans, as well as between the different stress studies almost impossible. In contrast, rodent models of chronic psychosocial stress represent situations more akin to those faced by humans and consequently seem to hold more clinical relevance. Our laboratory has developed a model in which mice are exposed to social stress for 19 continuous days, namely the chronic subordinate colony housing (CSC) paradigm, to help bridge this gap. The main aim of the current review article is to provide a detailed summary of the behavioral, physiological, neuronal, and immunological consequences of the CSC paradigm, and wherever possible relate the findings to other stress models and to the human situation.

Repeated psychosocial stress at night, but not day, affects the central molecular clock

We have recently demonstrated that the outcome of repeated social defeat (SD) on behavior, physiology and immunology is more negative when applied during the dark/active phase as compared with the light/inactive phase of male C57BL/6 mice. Here, we investigated the effects of the same stress paradigm, which combines a psychosocial and novelty stressor, on the circadian clock in transgenic PERIOD2::LUCIFERASE (PER2::LUC) and wildtype (WT) mice by subjecting them to repeated SD, either in the early light phase (social defeat lightu2009=u2009SDL) or in the early dark phase (social defeat darku2009=u2009SDD) across 19 days. The PER2::LUC rhythms and clock gene mRNA expression were analyzed in the suprachiasmatic nucleus (SCN) and the adrenal gland, and PER2 protein expression in the SCN was assessed. SDD mice showed increased PER2::LUC rhythm amplitude in the SCN, reduced Per2 and Cryptochrome1 mRNA expression in the adrenal gland, and increased PER2 protein expression in the posterior part of the SCN compared with single-housed control (SHC) and SDL mice. In contrast, PER2::LUC rhythms in the SCN of SDL mice were not affected. However, SDL mice exhibited a 2-hour phase advance of the PER2::LUC rhythm in the adrenal gland compared to SHC mice. Furthermore, plasma levels of brain-derived neurotrophic factor (BDNF) and BDNF mRNA in the SCN were elevated in SDL mice. Taken together, these results show that the SCN molecular rhythmicity is affected by repeated SDD, but not SDL, while the adrenal peripheral clock is influenced mainly by SDL. The observed increase in BDNF in the SDL group may act to protect against the negative consequences of repeated psychosocial stress.

Individual differences in stress vulnerability: The role of gut pathobionts in stress-induced colitis.

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.

HPA axis changes during the initial phase of psychosocial stressor exposure in male mice

Chronic subordinate colony (CSC) housing for 19 days results in unaffected basal morning corticosterone (CORT) levels despite a pronounced increase in adrenal mass, likely mediated by an attenuation of adrenal corticotropin (ACTH) responsiveness. Given that the pronounced increase in basal morning plasma CORT levels returns to baseline as early as 48u200ah after the start of CSC, it is likely that the attenuated ACTH responsiveness develops already during this initial phase. This was tested in the present study. In line with previous findings, basal morning plasma CORT levels were elevated following 10u200ah, but not 48u200ah, of CSC exposure. Basal morning plasma ACTH concentrations and relative in vivo adrenal CORT content were increased following 10u200ah and to a lesser extent following 48u200ah of CSC exposure, positively correlating. Relative in vitro adrenal CORT secretion in response to ACTH (100u200anM) and kidney protein expression of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) were unaffected following both time points. Adrenal mRNA expression of key steroidogenic enzymes was unaffected/decreased following 10u200ah and unaffected/increased following 48u200ah of CSC exposure. Together, our findings suggest that basal plasma hypercorticism during the initial CSC phase is mainly prevented by an attenuation of pituitary ACTH release. An increased absolute adrenal weight following 10u200ah, but not 48u200ah, of CSC exposure indicates that restoration of normal adrenal mass also adds to a lesser extent to prevent basal hypercorticism. A contributing role of alterations in enzymatic CORT degradation and steroidogenic enzyme availability is likely, but has to be further addressed in future studies.

Less immune activation following social stress in rural vs. urban participants raised with regular or no animal contact, respectively

Significance Our results show that a standardized laboratory psychosocial stressor causes a greater inflammatory response in young healthy participants with an urban upbringing in the absence of pets, relative to young healthy participants with a rural upbringing in the presence of farm animals. In view of the known links between persistent inflammatory states and psychiatric disturbances, and considering that many stress-associated physical and mental disorders are more prevalent in environments offering a narrow range of microbial exposures, we feel that our findings are of general interest and significance. Moreover, we feel our study is timely, as urbanization and the associated socioeconomic consequences are increasing. Urbanization is on the rise, and environments offering a narrow range of microbial exposures are linked to an increased prevalence of both physical and mental disorders. Human and animal studies suggest that an overreactive immune system not only accompanies stress-associated disorders but might even be causally involved in their pathogenesis. Here, we show in young [mean age, years (SD): rural, 25.1 (0.78); urban, 24.5 (0.88)] healthy human volunteers that urban upbringing in the absence of pets (n = 20), relative to rural upbringing in the presence of farm animals (n = 20), was associated with a more pronounced increase in the number of peripheral blood mononuclear cells (PBMCs) and plasma interleukin 6 (IL-6) concentrations following acute psychosocial stress induced by the Trier social stress test (TSST). Moreover, ex vivo-cultured PBMCs from urban participants raised in the absence of animals secreted more IL-6 in response to the T cell-specific mitogen Con A. In turn, antiinflammatory IL-10 secretion was suppressed following TSST in urban participants raised in the absence of animals, suggesting immunoregulatory deficits, relative to rural participants raised in the presence of animals. Questionnaires, plasma cortisol, and salivary α-amylase, however, indicated the experimental protocol was more stressful and anxiogenic for rural participants raised in the presence of animals. Together, our findings support the hypothesis that urban vs. rural upbringing in the absence or presence of animals, respectively, increases vulnerability to stress-associated physical and mental disorders by compromising adequate resolution of systemic immune activation following social stress and, in turn, aggravating stress-associated systemic immune activation.

Blocking metabotropic glutamate receptor subtype 5 relieves maladaptive chronic stress consequences.

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.

Environmental factors substantially affect the outcome of an established murine model for chronic psychosocial stress

Chronic subordinate colony housing (CSC, 19 days, 12 h/12 h light/dark cycle (LDC), normal drinking water (NW)), an established model for chronic psychosocial stress in male mice, has been repeatedly shown to cause basal evening hypocorticism, spontaneous colitis and increased anxiety-related behavior. However, running this model at a novel facility under slightly different environmental conditions (14 h/10 h LDC, acidified drinking water (AW)) revealed that both, the physiology of single housed control (SHC) mice as well as the extremely robust CSC effects are strongly dependent on the prevailing environmental conditions. Notably, a 14 h/10 h LDC and AW represent standard housing conditions in many animal facilities, whereas commercial animal suppliers (e.g. Charles River, Sulzfeld, Germany) often employ a 12 h/12 h LDC and NW. In detail, our data indicate that SHC mice kept under a 14 h/10 h LDC and AW, even 4 weeks after delivery from Charles River show increased basal morning plasma corticosterone (CORT) levels. This effect was even more pronounced two weeks after arrival and could be completely prevented by employing a 12 h/12 h LDC. Importantly, using a 12 h/12 h LDC, but keeping an AW, did not bring back any of the above described CSC effects. However, the combination of a 12 h/12 h LDC and NW, instead of AW, at least brought back the well-known effects of CSC on HPA axis functionality, namely basal evening hypocorticism and a reduced adrenal in vitro ACTH sensitivity. Given that CSC exposure under these conditions still does not cause anxiety and colitis, we are currently investigating other environmental factors that might play a role.

121. Chronic psychosocial stress promotes tumor growth by the mobilization of MDSC into peripheral organs

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 19xa0days 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.