Mucosal immunosuppression and epithelial barrier defects are key events in murine psychosocial stress-induced colitis
Highlights
► 10 h of psychosocial stress cause local colonic immunosuppression mediated by adrenal hormones. ► 10 h of psychosocial stress result in a reduction of colonic barrier functions. ► 10 h of psychosocial stress cause proliferation and translocation of commensal bacteria. ► Translocation of comensal bacteria is causal for the initiation of stress-induced colitis. ► Immune-modulatory effect of adrenal hormones is essential for development of full blown colitis.
Introduction
Inflammatory disorders, such as Crohn’s disease (CD) and ulcerative colitis (UC) represent a major health concern, particularly in Western societies, with a life time prevalence of approximately 0.1% (Singh et al., 2001). Despite this, the mechanisms underlying the development of these disorders, particularly those that initiate the disease, remain poorly understood. However, a number of risk factors are known, including chronic psychosocial stress exposure (Bernstein et al., 2010, Duffy et al., 1991, Salem and Shubair, 1967), and we recently demonstrated that 19 days of chronic subordinate colony housing (CSC) causes spontaneous (Reber et al., 2007) and aggravates chemically-induced (Reber et al., 2008) colitis in male mice. The CSC-induced spontaneous colitis is reflected by an increase in the histological damage score (Reber et al., 2007), in the cytokine secretion from isolated lamina propria mononuclear cells (LPMC) and mesenteric lymph node cells (mesLNC), in the number of colonic macrophages, dendritic cells, and T helper cells, and colonic IFN-γ and TNF mRNA expression (Supplementary Fig. 1 and (Reber et al., 2007, Reber et al., 2008)).
The hyperactive immune system following 19 days of CSC exposure is driven by the combination of low plasma glucocorticoid (GC) levels due to adrenal insufficiency, and GC resistance, which both occur during the later stages of chronic psychosocial stressor exposure (Reber et al., 2007). However, an initial stress-induced rise in GC during the first 10 h of CSC exposure (Reber et al., 2007) (see also Supplementary Fig. 2) seems critically involved in these pathological adaptations, as adrenalectomy (ADX) prior to CSC ameliorates cytokine secretion from isolated mesLNC and completely prevents the development of histological abnormalities (Reber et al., 2007, Reber et al., 2008). As ADX not only removes circulating GC but also mineralocorticoids and catecholamines, it can not be 100% excluded that adrenal hormones different from corticosterone at least partly are also involved in this initial pro-inflammatory effect of the adrenal glands.
Therefore, to confirm an initial local immunosuppression by high GC levels (Dhabhar and McEwen, 2001, Dhabhar and McEwen, 1997, Dhabhar and McEwen, 1999), we compared the immune status at the colonic tissue of CSC and single housed control (SHC) mice. High GC levels also result in increased colonic permeability (for review see Soderholm and Perdue, 2001), thus we characterized colonic-barrier functions during the initial 10 h of CSC. ADX prior to CSC should thereby dissect the causal involvement of adrenal hormones, most likely GC, in the development of barrier and immune defects during the initial phase of chronic stress. Prolonged stressor exposure increased the number of commensal gut bacteria in stool samples collected from the large intestine (Bailey et al., 2010) and a stress-induced increase in epithelial permeability of the gastrointestinal tract promotes bacterial translocation into various organs and body compartments (Ando et al., 2000, Bailey et al., 2006, Ding et al., 2004, Everson and Toth, 2000, Wang et al., 2004). Furthermore, suppression of the colonic immune system by excessive GC might result in unhindered proliferation of luminal, as well as translocated, bacteria of the commensal flora. Therefore, we assessed the bacterial load in different tissues during the initial phase of CSC. High levels of colonic antigens during the initial phase of CSC are likely to be a prerequisite for the subsequent development of spontaneous colitis. According to our hypothesis, during later stages of chronic psychosocial stress, when there is a lack of immunosuppressive GC signalling, mediated via both GC resistance (Reber et al., 2007, Schmidt et al., 2010) and hypocorticism (Reber et al., 2007, Reber and Neumann, 2008, Schmidt et al., 2010, Veenema et al., 2008), the over-reactive immune system targets excessive colonic antigens and consequently promotes the inflammatory processes. Accordingly, we investigated the causal involvement of intestinal bacteria in the development of CSC-induced colitis using prolonged treatment with broad-spectrum antibiotics prior to, and during 19 days of CSC exposure.
Together, the overall aim of the study was to identify relevant mechanisms that underlie stress-induced initiation of colitis by dissecting the early interplay between the endocrine system and the colonic immune and barrier functions.
Section snippets
Animals
As previously described (Reber et al., 2007, Reber et al., 2008, Reber and Neumann, 2008, Schmidt et al., 2010, Singewald et al., 2009, Veenema et al., 2008) male C57BL/6 mice (Charles River, Sulzfeld, Germany) weighing 19–22 g (experimental mice) or 30–35 g (used as residents during the CSC housing) were used in the current study.
Experimental procedure
Immune and barrier functions and the number of colony forming units (CFU) in stool, colonic and mesenteric lymph node tissue, and plasma corticosterone were assessed following 10 h of CSC. In another set of mice ADX was performed 2 weeks prior to the start of a 10-h CSC exposure. Finally, experimental mice were treated with four different antibiotics (see below) during the 4 weeks before as well as during subsequent 19 days CSC exposure. Another set of mice was killed following 19 days of CSC for
Immunological changes following 10 h of CSC
In contrast to 19 days of CSC resulting in attenuated GC-mediated immuno-suppression (Reber et al., 2007, Schmidt et al., 2010) and the development of spontaneous colitis (Reber et al., 2007) (see also Supplementary Fig. 1), acute stressor exposure should have the opposite effect. Therefore, we investigated whether a suppression of the local colonic immune system was observed after 10 h CSC. In support of this hypothesis, the numbers of F4/80+ macrophages (p = 0.050), CD11c+ dendritic cells (p =
Discussion
Our findings reveal the initial mechanisms, which underlie chronic psychosocial stress-induced colitis: namely increased bacterial load in the stool and colonic tissue, rendered possible by a profound adrenal hormone-driven immune suppression and a concurrent deterioration in epithelial barrier functions. Together with what is already known these data indicate that translocation of commensal bacteria is crucial in the initiation of chronic stress-induced colonic inflammation. Moreover, they
Conflict of interest
All authors declare that there are no conflicts of interest.
Acknowledgments
The authors are grateful to N. Grunwald, N. Beitelrock, and N. Uschold (all from the Department of Behavioural and Molecular Neuroendocrinology) and to N. Dunger (Department of Internal Medicine I) for excellent technical assistance, and to Dr. F. Bataille and S. Gaak (Department of Pathology, University Clinic Regensburg) and Dr. H.J. Linde (Department of Medical Microbiology, University Clinic Regensburg) for their help with the quantification of epithelial cell proliferation and performing
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