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Inflammatory conditions, including allergic asthma and conditions in which chronic low-grade inflammation is a risk factor, such as stress-related psychiatric disorders, are prevalent and are a significant cause of disability worldwide. Novel approaches for the prevention and treatment of these disorders are needed. One approach is the use of immunoregulatory microorganisms, such as Mycobacterium vaccae NCTC 11659, which have anti-inflammatory, immunoregulatory, and stress-resilience properties. However, little is known about how M. vaccae NCTC 11659 affects specific immune cell targets, including monocytes, which can traffic to peripheral organs and the central nervous system and differentiate into monocyte-derived macrophages that, in turn, can drive inflammation and neuroinflammation. In this study, we investigated the effects of M. vaccae NCTC 11659 and subsequent lipopolysaccharide (LPS) challenge on gene expression in human monocyte-derived macrophages. THP-1 monocytes were differentiated into macrophages, exposed to M. vaccae NCTC 11659 (0, 10, 30, 100, 300 µg/mL), then, 24 h later, challenged with LPS (0, 0.5, 2.5, 250 ng/mL), and assessed for gene expression 24 h following challenge with LPS. Exposure to M. vaccae NCTC 11659 prior to challenge with higher concentrations of LPS (250 ng/mL) polarized human monocyte-derived macrophages with decreased IL12A, IL12B, and IL23A expression relative to IL10 and TGFB1 mRNA expression. These data identify human monocyte-derived macrophages as a direct target of M. vaccae NCTC 11659 and support the development of M. vaccae NCTC 11659 as a potential intervention to prevent stress-induced inflammation and neuroinflammation implicated in the etiology and pathophysiology of inflammatory conditions and stress-related psychiatric disorders.

Purpose Endochondral ossification, which involves transdifferentiation of chondrocytes into osteoblasts, is an important process involved in the development and postnatal growth of most vertebrate bones as well as in bone fracture healing. To study the basic molecular mechanisms of this process, a robust and easy-to-use in vitro model is desirable. Therefore, we aimed to develop a standardized in vitro assay for the transdifferentiation of chondrogenic cells towards the osteogenic lineage. Methods Murine chondrogenic ATDC5 cells were differentiated into the chondrogenic lineage for seven days and subsequently differentiated towards the osteogenic direction. Gene expression analysis of pluripotency, as well as chondrogenic and osteogenic markers, cell–matrix staining, and immunofluorescent staining, were performed to assess the differentiation. In addition, the effects of Wnt3a and lipopolysaccharides (LPS) on the transdifferentiation were tested by their addition to the osteogenic differentiation medium. Results Following osteogenic differentiation, chondrogenically pe-differentiated cells displayed the expression of pluripotency and osteogenic marker genes as well as alkaline phosphatase activity and a mineralized matrix. Co-expression of Col2a1 and Col1a1 after one day of osteogenic differentiation indicated that osteogenic cells had differentiated from chondrogenic cells. Wnt3a increased and LPS decreased transdifferentiation towards the osteogenic lineage. Conclusion We successfully established a rapid, standardized in vitro assay for the transdifferentiation of chondrogenic cells into osteogenic cells, which is suitable for testing the effects of different compounds on this cellular process.

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.

ZUSAMMENFASSUNGEs gibt zahlreiche Hinweise darauf, dass chronische negative Erfahrungen, insbesondere chronischer psychosozialer Stress, einen wesentlichen Risikofaktor für die Entstehung vieler somatischer und affektiver Störungen darstellen, einschließlich chronischer entzündlicher Darmerkrankungen (CED) und der posttraumatischen Belastungsstörung (PTBS). Die Mechanismen, die der Entstehung chronischer stressbedingter Erkrankungen zugrunde liegen, sind jedoch noch weitgehend unbekannt und die derzeitigen Behandlungs- und Präventionsstrategien sind unzureichend wirksam und zuverlässig. Ein besseres Verständnis der Mechanismen, die an der Entstehung und Aufrechterhaltung chronischer stressbedingter Erkrankungen beteiligt sind, kann zu neuen Ansätzen in der Prävention und Behandlung dieser Erkrankungen führen. In diesem 2-teiligen Review fassen wir die theoretischen Grundlagen (Teil 1) sowie die zugrunde liegenden Mechanismen (Teil 2) zusammen, welche die Hypothese stützen, dass ein Anstieg der Immun(re)aktivität und des Entzündungsgeschehens in der heutigen modernen Gesellschaft, potenziell gefördert durch einen verminderten Kontakt mit immunregulatorischen Mikroorganismen („Old Friends“), ursächlich an der gesteigerten Anfälligkeit für stressbedingte Pathologien sein könnte. Darüber hinaus diskutieren wir den künstlich herbeigeführten Kontakt mit Old Friends als vielversprechende Strategie zur Förderung der Stressresilienz und zur Vorbeugung/Behandlung chronischer stressbedingter Erkrankungen.

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 psychosocial stress adversely affects human morbidity and is a risk factor for inflammatory disorders, liver diseases, obesity, metabolic syndrome, and major depressive disorder (MDD). In recent studies, we found an association of MDD with an increase of acid sphingomyelinase (ASM) activity. Thus, we asked whether chronic psychosocial stress as a detrimental factor contributing to the emergence of MDD would also affect ASM activity and sphingolipid (SL) metabolism. To induce chronic psychosocial stress in male mice we employed the chronic subordinate colony housing (CSC) paradigm and compared them to non-stressed single housed control (SHC) mice. We determined Asm activity in liver and serum, hepatic SL concentrations as well as hepatic mRNA expression of genes involved in SL metabolism. We found that hepatic Asm activity was increased by 28% (P = 0.006) and secretory Asm activity by 47% (P = 0.002) in stressed mice. C16:0-Cer was increased by 40% (P = 0.008). Gene expression analysis further revealed an increased expression of tumor necrosis factor (TNF)-alpha (P = 0.009) and of several genes involved in SL metabolism (Cers5, P = 0.028; Cers6, P = 0.045; Gba, P = 0.049; Gba2, P = 0.030; Ormdl2, P = 0.034; Smpdl3B; P = 0.013). Our data thus provides first evidence that chronic psychosocial stress, at least in mice, induces alterations in SL metabolism, which in turn might be involved in mediating the adverse health effects of chronic psychosocial stress and peripheral changes occurring in mood disorders., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe; 1120

The neuropeptide oxytocin (OXT) has generated considerable interest as potential treatment for psychiatric disorders, including anxiety and autism spectrum disorders. However, the behavioral and molecular consequences associated with chronic OXT treatment and chronic receptor (OXTR) activation have scarcely been studied, despite the potential therapeutic long-term use of intranasal OXT. Here, we reveal that chronic OXT treatment over two weeks increased anxiety-like behavior in rats, with higher sensitivity in females, contrasting the well-known anxiolytic effect of acute OXT. The increase in anxiety was transient and waned 5 days after the infusion has ended. The behavioral effects of chronic OXT were paralleled by activation of an intracellular signaling pathway, which ultimately led to alternative splicing of hypothalamic corticotropin-releasing factor receptor 2α (Crfr2α), an important modulator of anxiety. In detail, chronic OXT shifted the splicing ratio from the anxiolytic membrane-bound (mCRFR2α) form of CRFR2α towards the soluble CRFR2α (sCRFR2α) form. Experimental induction of alternative splicing mimicked the anxiogenic effects of chronic OXT, while sCRFR2α-knock down reduced anxiety-related behavior of male rats. Furthermore, chronic OXT treatment triggered the release of sCRFR2α into the cerebrospinal fluid with sCRFR2α levels positively correlating with anxiety-like behavior. In summary, we revealed that the shifted splicing ratio towards expression of the anxiogenic sCRFR2α underlies the adverse effects of chronic OXT treatment on anxiety.

Severe injuries are frequently accompanied by hemorrhagic shock and harbor an increased risk for complications. Local or systemic inflammation after trauma/hemorrhage may lead to a leaky intestinal epithelial barrier and subsequent translocation of gut microbiota, potentially worsening outcomes. To evaluate the extent with which trauma affects the gut microbiota composition, we performed a post hoc analysis of a murine model of polytrauma and hemorrhage. Four hours after injury, organs and plasma samples were collected, and the diversity and composition of the cecal microbiome were evaluated using 16S rRNA gene sequencing. Although cecal microbial alpha diversity and microbial community composition were not found to be different between experimental groups, norepinephrine support in shock animals resulted in increased alpha diversity, as indicated by higher numbers of distinct microbial features. We observed that the concentrations of proinflammatory mediators in plasma and intestinal tissue were associated with measures of microbial alpha and beta diversity and the presence of specific microbial drivers of inflammation, suggesting that the composition of the gut microbiome at the time of trauma, or shortly after trauma exposure, may play an important role in determining physiological outcomes. In conclusion, we found associations between measures of gut microbial alpha and beta diversity and the severity of systemic and local gut inflammation. Furthermore, our data suggest that four hours following injury is too early for development of global changes in the alpha diversity or community composition of the intestinal microbiome. Future investigations with increased temporal-spatial resolution are needed in order to fully elucidate the effects of trauma and shock on the gut microbiome, biological signatures of inflammation, and proximal and distal outcomes.

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

There is convincing evidence from different mouse models that chronic psychosocial stress promotes splenomegaly, basal and lipopolysaccharide (LPS)-induced in vitro splenocyte activation and insensitivity towards glucocorticoids (GC) in in vitro LPS-treated splenocytes. However, we just recently showed, employing the chronic subordinate colony housing (CSC) paradigm, that bite wounds received during stressor exposure drive these stress-induced spleen changes. As skin wounds induced by planned surgery or physical trauma are more adequately reflecting what chronically stressed humans are likely to experience, it was the objective of the present study to investigate whether abdominal surgery prior to stressor exposure also promotes respective stress-induced spleen effects in the absence of any bite wounds. In line with our hypothesis, abdominal surgery prior to CSC induced splenomegaly, increased in vitro cell viability under basal and LPS conditions as well as the delta response to LPS (LPS – basal), and promoted the inability of isolated splenocytes to respond with a decreased cell viability to increasing concentrations of corticosterone following LPS-stimulation in vitro. Together with previous data, these findings demonstrate that physical injury, either in form of received bite wounds during stressor exposure or in form of abdominal surgery prior to stressor exposure, promotes the development of splenic immune activation and GC resistance.

There is a considerable body of evidence indicating that chronic adverse experience, especially chronic psychosocial stress/trauma, represents a major risk factor for the development of many somatic and affective disorders, including inflammatory bowel disease (IBD) and posttraumatic stress disorder (PTSD). However, the mechanisms underlying the development of chronic stress-associated disorders are still in large part unknown, and current treatment and prevention strategies lack efficacy and reliability. A greater understanding of mechanisms involved in the development and persistence of chronic stress-induced disorders may lead to novel approaches to prevention and treatment of these disorders. In this review, we provide evidence indicating that increases in immune (re-)activity and inflammation, potentially promoted by a reduced exposure to immunoregulatory microorganisms ("Old Friends") in today's modern society, may be causal factors in mediating the vulnerability to development and persistence of stress-related pathologies. Moreover, we discuss strategies to increase immunoregulatory processes and attenuate inflammation, as for instance contact with immunoregulatory Old Friends, which appears to be a promising strategy to promote stress resilience and to prevent/treat chronic stress-related disorders.

Dysregulated intestinal epithelial apoptosis initiates gut injury, alters the intestinal barrier, and can facilitate bacterial translocation leading to a systemic inflammatory response syndrome (SIRS) and/or multi-organ dysfunction syndrome (MODS). A variety of gastrointestinal disorders, including inflammatory bowel disease, have been linked to intestinal apoptosis. Similarly, intestinal hyperpermeability and gut failure occur in critically ill patients, putting the gut at the center of SIRS pathology. Regulation of apoptosis and immune-modulatory functions have been ascribed to Thirty-eight-negative kinase 1 (TNK1), whose activity is regulated merely by expression. We investigated the effect of TNK1 on intestinal integrity and its role in MODS. TNK1 expression induced crypt-specific apoptosis, leading to bacterial translocation, subsequent septic shock, and early death. Mechanistically, TNK1 expression in vivo resulted in STAT3 phosphorylation, nuclear translocation of p65, and release of IL-6 and TNF-alpha. A TNF-alpha neutralizing antibody partially blocked development of intestinal damage. Conversely, gut-specific deletion of TNK1 protected the intestinal mucosa from experimental colitis and prevented cytokine release in the gut. Finally, TNK1 was found to be deregulated in the gut in murine and porcine trauma models and human inflammatory bowel disease. Thus, TNK1 might be a target during MODS to prevent damage in several organs, notably the gut.

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.

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.

Background Short non-coding microRNAs (miRNAs) are involved in various cellular processes during disease progression of Crohn’s disease (CD) and remarkably stable in feces, which make them attractive biomarker candidates for reflecting intestinal inflammatory processes. Here we investigated the potential of fecal miRNAs as noninvasive and translational CD biomarkers. Methods MiRNAs were screened in feces of 52 patients with CD and 15 healthy controls using RNA sequencing and the results were confirmed by PCR. The relationship between fecal miRNA levels and the clinical CD activity index (CDAI) or CD endoscopic index of severity (CDEIS) was explored, respectively. Additionally, fecal miRNAs were investigated in dextran sodium sulfate, adoptive T-cell transfer, and Helicobacter typhlonius/stress-induced murine colitis models using the NanoString platform. Results Nine miRNAs (miR-15a-5p, miR-16-5p, miR-128-3p, miR-142-5p, miR-24-3p, miR-27a-3p, miR-223-3p, miR-223-5p, and miR-3074-5p) were significantly (adj. P < 0.05, >3-fold) increased whereas 8 miRNAs (miR-10a-5p, miR-10b-5p, miR-141-3p, miR-192-5p, miR-200a-3p, miR-375, miR-378a-3p, and let-7g-5p) were significantly decreased in CD. MiR-192-5p, miR-375, and miR-141-3p correlated (P < 0.05) with both CDAI and CDEIS whereas miR-15a-5p correlated only with CDEIS. Deregulated expression of miR-223-3p, miR-16-5p, miR-15a-5p, miR-24-3p, and miR-200a-3p was also observed in murine models. The identified altered fecal miRNA levels reflect pathophysiological mechanisms in CD, such as Th1 and Th17 inflammation, autophagy, and fibrotic processes. Conclusions Our translational study assessed global fecal miRNA changes of patients with CD and relevant preclinical models. These fecal miRNAs show promise as translational and clinically useful noninvasive biomarkers for mechanistic investigation of intestinal pathophysiology, including monitoring of disease progression., Lay Summary MicroRNAs are involved in disease progression of Crohn’s disease (CD). This study identified significantly altered fecal levels of 17 microRNAs in CD compared to healthy subjects. These microRNAs reflect pathophysiological mechanisms and show promise as clinically useful, noninvasive CD biomarkers.

It has been extensively studied in several mouse models how chronic, in particular chronic psychosocial, stressors facilitate the (re)activity of the innate immune system and, consequently, drive stress-associated pathologies. Here we first summarize the resulting concept and underlying mechanisms, proposing that social stress-induced bone marrow myelopoiesis, priming, emigration and activation of newly formed myeloid cells and accumulation of these cells in the spleen, gut, brain and fracture hematoma promote septic shock, colitis, anxiety and disturbed fracture healing, respectively. We further propose and discuss the hypothesis that it is not the social character of a particular stressor that promotes splenic invasion and subsequent full activation of stress-induced myeloid cells, but rather the occurrence of bite wounds as a result of direct physical interaction. Finally, we discuss the hypothesis that it is the combination of chronic stress, regardless of whether social or non-social in nature, and any kind of planned (i.e. surgery) or unplanned (i.e. bite wounds, injury) physical trauma that drives splenic invasion and subsequent full activation of stress-induced myeloid cells.

Chronic psychosocial stress/trauma represents an increasing burden in our modern society and a risk factor for the development of mental disorders, including posttraumatic stress disorder (PTSD). PTSD, in turn, is highly comorbid with a plethora of inflammatory disorders and has been associated with increased bone fracture risk. Since a balanced inflammatory response after fracture is crucial for successful bone healing, we hypothesize that stress/trauma alters the inflammatory response after fracture and, consequently, compromises fracture healing. Here we show, employing the chronic subordinate colony housing (CSC) paradigm as a clinically relevant mouse model for PTSD, that mice subjected to CSC displayed increased numbers of neutrophils in the early fracture hematoma, whereas T lymphocytes and markers for cartilage-to-bone transition and angiogenesis were reduced. At late stages of fracture healing, CSC mice were characterized by decreased bending stiffness and bony bridging of the fracture callus. Strikingly, a single systemic administration of the β-adrenoreceptor (AR) blocker propranolol before femur osteotomy prevented bone marrow mobilization of neutrophils and invasion of neutrophils into the fracture hematoma, both seen in the early phase after fracture, as well as a compromised fracture healing in CSC mice. We conclude that chronic psychosocial stress leads to an imbalanced immune response after fracture via β-AR signaling, accompanied by disturbed fracture healing. These findings offer possibilities for clinical translation in patients suffering from PTSD and fracture.

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.

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.

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.

Background Hypoxemia and tissue ischemia during hemorrhage as well as formation of oxygen and nitrogen radicals during resuscitation promote hyperinflammation and, consequently, trigger severe multi-organ failure (MOF). Individuals diagnosed with stress-related disorders or reporting a life history of psychosocial stress are characterized by chronic low-grade inflammation and a reduced glucocorticoid (GC) signaling. We hypothesized that exposure to chronic psychosocial stress during adulthood prior to hemorrhagic shock increases oxidative/nitrosative stress and therefore the risk of developing MOF in mice. Methods and findings To induce chronic psychosocial stress linked to mild immune activation and reduced GC signaling in male mice, the chronic subordinate colony housing (CSC) paradigm was employed. Single-housed (SHC) mice were used as controls. Subsequently, CSC and SHC mice were exposed to hemorrhagic shock following resuscitation to investigate the effects of prior psychosocial stress load on survival, organ function, metabolism, oxidative/nitrosative stress, and inflammatory readouts. An increased adrenal weight in CSC mice indicates that the stress paradigm reliably worked. However, no effect of prior psychosocial stress on outcome after subsequent hemorrhage and resuscitation could be detected. Conclusions Chronic psychosocial stress during adulthood is not sufficient to promote hemodynamic complications, organ dysfunction, metabolic disturbances and did not increase the risk of MOF after subsequent hemorrhage and resuscitation. Intravenous norepinephrine to keep target hemodynamics might have led to a certain level of oxidative stress in both groups and, therefore, disguised potential effects of chronic psychosocial stress on organ function after hemorrhagic shock in the present murine trauma model.