The contributing role of the intestinal microbiota in stressor-induced increases in susceptibility to enteric infection and systemic immunomodulation

Hormones and Behavior 62 (2012) 286–294

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Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh

Review

The contributing role of the intestinal microbiota in stressor-induced increases in susceptibility to enteric infection and systemic immunomodulation Michael T. Bailey ⁎ Institute for Behavioral Medicine Research, College of Medicine, The Ohio State University, Columbus, OH 43210, USA Division of Oral Biology, College of Dentistry, The Ohio State University, Columbus, OH 43210, USA Center for Microbial Interface Biology, College of Medicine, The Ohio State University, Columbus, OH 43210, USA

a r t i c l e

i n f o

a b s t r a c t

Available online 15 February 2012

This article is part of a Special Issue “Neuroendocrine-Immune Axis in Health and Disease.”

Keywords: Stress Microbiota Citrobacter rodentium Macrophage Microbial killing Peroxynitrite Mast cell Intestinal epithelial cell

The body is colonized by highly complex and genetically diverse communities of microbes, the majority of which reside within the intestines in largely stable but dynamically interactive climax communities. These microbes, referred to as the microbiota, have many functions that enhance the health of the host, and it is now recognized that the microbiota influence both mucosal and systemic immunity. The studies outlined in this review demonstrate that the microbiota are also involved in stressor-induced immunomodulation. Exposure to different types of stressors, including both physical and psychological stressors, changes the composition of the intestinal microbiota. The altered profile increases susceptibility to an enteric pathogen, i.e., Citrobacter rodentium, upon oral challenge, but is also associated with stressor-induced increases in innate immune activity. Studies using germfree mice, as well as antibiotic-treated mice, provide further evidence that the microbiota contribute to stressor-induced immunomodulation; stressor-induced increases in splenic macrophage microbicidal activity fail to occur in mice with no, or reduced, intestinal microbiota. While the mechanisms by which microbiota can impact mucosal immunity have been studied, how the microbiota impact systemic immune responses is not clear. A mechanism is proposed in which stressor-induced degranulation of mucosal mast cells increases the permeability of the intestines. This increased permeability would allow intact bacteria and/or bacterial products (like peptidoglycan) to translocate from the lumen of the intestines to the interior of the body, where they directly, or indirectly, prime the innate immune system for enhanced reactivity to antigenic stimulation. © 2012 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The intestinal microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of the intestinal microbiota . . . . . . . . . . . . . . . . . . . . . . . Function of the intestinal microbiota . . . . . . . . . . . . . . . . . . . . . . . . Impact of stressor exposure on the structure and function of the microbiota . . . . . . . Culture-based studies in laboratory rodents, non-human primates, and humans . . . Rodent studies involving prolonged/chronic stressors . . . . . . . . . . . . . . . . Effects on gut microbiota community structure. . . . . . . . . . . . . . . . Effects on gut microbiota function. . . . . . . . . . . . . . . . . . . . . . Studies involving the social disruption stressor . . . . . . . . . . . . . . . . . . . Effects on gut microbiota community structure. . . . . . . . . . . . . . . . Effects on gut microbiota function. . . . . . . . . . . . . . . . . . . . . . Importance of mast cells in linking the microbiota to stressor-induced immunomodulation Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ The Ohio State University, Institute for Behavioral Medicine Research, College of Medicine, 257 IBMR Building, 460 Medical Center Dr., Columbus, OH 43210, USA. E-mail address: [email protected]. 0018-506X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2012.02.006

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Introduction The body is colonized by an enormous array of microbes collectively referred to as the microbiota. All surfaces of the body (i.e., the oral cavity, skin, reproductive tract, respiratory tract, and gastrointestinal tract) are colonized by all known forms of microbes (i.e., bacteria, archaea, protists, as well as viruses), but the majority of the microbiota are bacteria that reside within the gastrointestinal tract. The microbiota are thought to outnumber cells of the body by a factor of 10 (i.e., approximately 1 × 10 14 bacteria: 1 × 10 13 human cells). Not only are there a large number of bacteria residing on/within the body, but there is also a large diversity of bacterial types. Estimates of bacterial diversity vary widely (e.g., ranging from a few hundred to over 30,000 different bacterial types). But, most estimates indicate that between 500 and 1000 different bacterial species reside as the microbiota (Xu and Gordon, 2003). The largest density of microbiota are found in the colon where they reside as stable climax communities as a result of ecological successions involving the selection of microbes best adapted for their given niche (Huffnagle, 2010). These climax communities are relatively resistant to change (Allison and Martiny, 2008), but many factors, including diet, antibiotic use, or exposure to different types of stressors, can cause transient alterations in the microbial communities (Antonopoulos et al., 2009; Bailey et al., 2010, 2011; Dethlefsen et al., 2008; Turnbaugh et al., 2008). While stable microbial communities confer health benefits to the host, dysbiosis (i.e., disruptions to the community structure) has been associated with negative health outcomes. The purpose of this review is to provide a general description of the intestinal microbiota and the impact that the stress response can have on community structure. The implications of stressor-induced alterations in the microbiota for stressor-induced increases in susceptibility to enteric infection and for stressor-induced immunomodulation will also be discussed.

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rDNA amplicons collected from 3 human participants demonstrated considerable variability. Approximately 130 bacterial types were identified in one participant, approximately 300 in the second participant, and approximately 200 in the third participant (Eckburg et al., 2005). The types of bacteria that were identified differed depending upon sampling location, i.e., samples collected from the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, rectum, or stool contained different bacterial communities. But, location of sample collection was less variable than inter-individual variability. For example, the bacterial community structures in the cecum and the stool from participant A were found to be similar. But, the bacterial communities in the cecum of participant A and the cecum of participant B were found to be distinct, thus demonstrating the large inter-individual variability (Eckburg et al., 2005). Despite the large inter-individual differences in predominant bacterial genera, the relative proportions of bacterial genera can be reflective of host factors. For example, in a recent study, investigators found that microbial communities from human stool could be classified into two groups based upon the relative abundance of bacteria in the genus Bacteroides or in the genus Prevotella. And, the relative abundance of bacteria in these two genera was predictive of whether their human hosts consumed carbohydrate-rich vs. fat-rich diets (Wu et al., 2011). Of course, Bacteroides and Prevotella are not the only microbes that reside within the colon, and other bacterial types, such as bacteria in the genus Bifidobacterium (in humans), Clostridium, and Eubacterium predominate in colons of mice and humans (Bailey et al., 2010, 2011; Sekirov et al., 2010). Aerobic organisms are less abundant in the intestines, but bacteria in the genera Escherichia, Enterococcus, and Streptococcus are often present. Importantly for this review, both humans and mice contain stable populations of bacteria in the genus Lactobacillus (Frese et al., 2011), which can have beneficial effects in the intestines. Function of the intestinal microbiota

The intestinal microbiota Structure of the intestinal microbiota All sections of the gastrointestinal tract are colonized by microbes, but the density of microbes increases from the proximal to distal intestinal segments. The colon harbors the most abundant and most diverse bacterial communities. It has long been a goal to determine which types of bacteria can reside within the colon, but previous studies have been hampered by the reliance on culture-based methods. The development and widespread use of culture-independent deep sequencing methodologies, such as pyrosequencing using the 454 FLX-Titanium platform (by Life Sciences), has dramatically increased our understanding of the diversity of the intestinal microbiota and of the structure of their communities. These ecology-based measures help to describe the composition of communities, with structure reflecting the overall composition of the community and the abundance of its individual members. Diversity encompasses both the richness (i.e., number of types of bacteria in a community) and evenness (i.e., the distribution of individual bacterial types). The composition of the microbiota can be predictive of the function of the microbiota, which refers to a community's activity (Robinson et al., 2010). The majority of colonic bacteria belong to either the phylum Firmicutes or the phylum Bacteroidetes, with a small proportion of bacteria residing within the Actinobacteria, Proteobacteria, Deferribacteres, TM7, Deinococcus, and Fusobacteria (Eckburg et al., 2005; Sekirov et al., 2010). This profile is found in most individuals and there is relatively low variability when bacteria are classified at the phylum level. But there is considerably more variability when bacteria are classified at lower taxonomic levels such as at the bacterial genus or species levels. For example, an early study involving the cloning and sequencing of approximately 13,000 microbial 16s

Studies have demonstrated that changes in community structure can result in changes to community function. While the structure of the microbial community functions to support the community itself (e.g., such as through nutrient flow from one community member to others), the intestinal microbiota serve multiple functions in host physiology (see (O'Hara and Shanahan, 2006) for a review). The microbiota contribute to the synthesis of many of our vitamins (such as vitamins K and B12) (Resta, 2009) and many components of the human metabolome are derived from microbes (Wikoff et al., 2009). Changes in the microbiota community structure can have adverse health consequences, and both nutrition/metabolic related disorders, like obesity (Ley et al., 2005; Turnbaugh et al., 2006, 2009), as well as immune-mediated diseases like the inflammatory bowel diseases (Frank et al., 2007, 2011; Ott et al., 2004; Walker et al., 2011) have been associated with altered profiles of intestinal bacteria. Studies from this laboratory have been heavily influenced by previous research demonstrating that the microbiota help to protect the host against pathogen colonization and invasion of mucosal surfaces, a phenomenon referred to as competitive exclusion. Ely Methnikoff hypothesized nearly 100 years ago that lactic acid bacteria that normally reside as part of the microbiota, such as bacteria in the genus Lactobacillus, were able to limit pathogen colonization and proliferation (Metchnikoff, 1908). It is now recognized that Lactobacillus spp., as well as other commensal bacteria, can prevent pathogen colonization of intestinal epithelial cells by creating a physical/chemical barrier against invading pathogens. Studies in vitro demonstrate that common members of the microbiota, like Lactobacillus acidophilus, Bifidobacterium breve, and B. infantis, can create physical barriers to prevent pathogens, such as E. coli, Yersinia pseudotuberculosis, and Salmonella enteritidis from invading host cells (Coconnier et al., 1993a, 1993b). Some members of the microbiota, such as L. acidophilus and

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L. reuteri can produce compounds that directly kill pathogenic bacteria, such as pathogenic E. coli and Salmonella spp. (Bernet-Camard et al., 1997; Eaton et al., 2011; Zhang et al., 2012). Likewise, certain bifidobacteria produce short chain fatty acids, like acetate, that can reduce the severity of hemorrhagic colitis induced by E. coli O157: H7 by neutralizing the pathogen-produced Shiga toxin (Fukuda et al., 2011). The protective effects of these microbes extend beyond just creating physical and chemical barriers to encroaching pathogens. Many members of the microbiota have been shown to significantly impact both mucosal, as well as systemic, immune responses either through direct effects of the microbiota on the immune cells themselves, or indirectly by altering the activity of intestinal epithelial cells that then influence the developing immune response. Although not widely studied, intestinal epithelial cells are likely crucial cells that link the microbiota to mucosal, as well as systemic, immunity (Rescigno, 2011). Intestinal epithelial cells provide a formidable barrier to intestinal microbes (Marchiando et al., 2010), and in order to cause disease, intestinal pathogens must either colonize intestinal epithelial cells, invade through these cells, or disrupt the tight junctions between them to invade the body. Such host–microbe interactions cause epithelial cells to produce inflammatory cytokines and chemokines, in part through the activation of transcription factors like NFκB, that trigger the mucosal immune response. Interestingly, regulation of NF-κB appears to be critical for both the maintenance of epithelial barrier integrity, as well as the development of overt colitis. For example, inhibition of NF-κB through conditional ablation of IκB kinase-γ, which is needed for NF-κB activation can result in apoptosis in colonic epithelial cells and severe chronic intestinal inflammation (Nenci et al., 2007), demonstrating that NFκB is important for maintaining intestinal homeostasis. However, NF-κB activation can also be pathologic; prolonged activation of NFκB can result in the over-production of inflammatory cytokines like TNF-α (Li and Verma, 2002; Neish, 2004). TNF-α is an integral component of the mucosal immune response to intestinal pathogens, but when produced in excess leads to tissue-damaging colitis (Zuo et al., 2010). This is evident in laboratory animals with experimental colitis, as well as human patients with inflammatory bowel disease (Kaser et al., 2010; Zuo et al., 2010). Strategies to reduce the NF-κB, or NF-κB-regulated inflammatory cytokines like TNF-α, are effective treatments for inflammatory bowel diseases (Kaser et al., 2010). Many types of microbiota have been shown to influence intestinal epithelial NF-κB activation. This has primarily been studied in the context of probiotics, which are live microorganisms that when administered in adequate amounts confer a health benefit on the host (Food and Agriculture Organization of the United Nations and World Health Organization, 2001). Most probiotics are isolates from the human body and typically reside as part of the microbiota. One important effect of some probiotic microbes is their ability to suppress colonic inflammatory responses. For example, members of the genus Lactobacillus can inhibit the translocation of NF-κB from the cytoplasm to the nucleus by inhibiting the ubiquitination and degradation of IκB, or by increasing the expression of negative regulators of pattern recognition receptor signaling (such as peroxisome proliferators-activated receptor (PPAR)-γ or single Ig IL-1 receptorrelated protein (SIGIRR)) (Artis, 2008; Shibolet and Podolsky, 2007). The ability of indigenous microbiota, as well as exogenous probiotics to reduce NF-κB activation in intestinal epithelial cells in turn limits the development of the intestinal inflammatory response (Rescigno, 2011). Thus, microbial influences on intestinal epithelial cells have the capacity to significantly modulate the developing mucosal immune response. In addition to effects on intestinal epithelial cells, many types of microbiota can directly suppress or enhance leukocyte functioning. For example, L. reuteri secretes an as yet unidentified factor(s) that suppresses TNF-α production by LPS-activated THP-1 monocytoid

cells (Jones and Versalovic, 2009). Likewise, the cell wall components of L. casei strain Shirota suppresses cytokine production in LPSactivated RAW264.7 macrophages (Yasuda et al., 2008). In contrast, extracellular polysaccharides derived from L. delbrueckii subspecies bulgaricus significantly increased the capacity of mouse splenocytes to produce cytokines (Makino et al., 2006). The effects of the microbiota on cells of the adaptive immune system have also been tested, and probiotic microbes, such as L. gasseri have been shown to suppress CD4+ T cell proliferation in vitro and prevent delayed type hypersensitivity reactions in vivo (Yoshida et al., 2011). Because of the impact that these microbes have on immune system activity, we have hypothesized that stressor-induced decreases in the natural populations of these microbes would in turn impact mucosal and systemic immunity. Impact of stressor exposure on the structure and function of the microbiota Culture-based studies in laboratory rodents, non-human primates, and humans It was recognized over 30 years ago that exposing laboratory animals to novel environments has a significant impact on the stability of the intestinal microbiota. In 1974, Tannock and Savage found that depriving mice of food, water, and bedding significantly decreased the number of lactobacilli that could be cultured from the stomach, small intestine, and large intestine, with the largest decrease found in the stomach (Tannock and Savage, 1974). Others have found that changes in environmental stimuli, such as chronic sleep deprivation, can cause a significant overgrowth of microbiota in the ileum and cecum (Everson and Toth, 2000). Fewer studies have focused on psychological stimuli, but an early study in cosmonauts demonstrated that the intestinal microbiota were significantly different during space flight (Lizko, 1987), with other studies suggesting that these effects could be due to the stressor of confinement (Holdeman et al., 1976). To further assess the effects of psychological stressors on the stability of the intestinal microbiota, bacteria were cultured from the stool of rhesus monkeys (Macaca mulatta) that were being separated from their mothers for husbandry purposes. The separation occurred after the monkeys had already self-weaned and were eating solid foods, but separation still induced a strong emotional reaction in the young monkeys. The levels of lactobacilli that could be cultured in the stool were significantly different the week following separation, with significantly lower levels of lactobacilli being found 3 days after the separation (Bailey and Coe, 1999). Although this reduction was not associated with stressor-induced increases in circulating cortisol, lactobacilli levels were inversely correlated with stressindicative behaviors. In general, monkeys that showed the strongest behavioral reaction to the maternal separation also had the lowest levels of lactobacilli. Interestingly, the decreased levels of lactobacilli tended to increase opportunistic infection with Campylobacter jejuni and Shigella flexneri, two intestinal pathogens that are endemic in monkey colonies (Bailey and Coe, 1999). This initial study suggested that a naturally occurring stressor changed the structure of the microbiota, manifest as a reduction in lactobacilli, and changed the function of the microbiota, manifest as a reduction in competitive exclusion. The effects of stressor exposure on lactobacilli have primarily been studied in laboratory animals, but one study found that stressor exposure reduced lactobacilli levels in humans (Knowles et al., 2008). Fecal lactobacilli levels were assessed in college students during a low stress period (i.e., the first week of the semester) and a high stress period (i.e., final exam week). The exam period was associated with significantly higher levels of perceived daily stress, and when compared to the low stress period, the levels of lactobacilli shed in the stool were significantly lower during the exam period (Knowles

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et al., 2008). Significant differences in diet also occurred across the two time periods, but given results from laboratory animals, it is likely that stressor-associated changes in human lactobacilli levels reflect an impact of the stressor as well as potential effects of diet. Rodent studies involving prolonged/chronic stressors Effects on gut microbiota community structure While stressor exposure decreases lactobacilli levels in both laboratory animals and humans, the effects of the stress response on other bacterial types have not been extensively studied. This led to an experiment in which culture-independent, pyrosequencing using the 454 FLX-Titanium platform was used to characterize the microbiota in mice exposed to a prolonged restraint stressor (Bailey et al., 2010). Prolonged restraint is a widely used murine stressor that has been extensively characterized in the literature and is the most commonly used murine stressor in biomedical and biobehavioral research (Buynitsky and Mostofsky, 2009). This stressor involves both a physical component (i.e., physical confinement) and a psychological component that is thought to reflect the animal's perception of burrow collapse and inescapability (Buynitsky and Mostofsky, 2009). Prolonged restraint reliably induces a physiological stress response that results in the elevation of endogenous corticosterone, epinephrine, and norepinephrine (Buynitsky and Mostofsky, 2009; Dobbs et al., 1993, 1996; Padgett et al., 1998). Thus, mice were exposed to the prolonged restraint [overnight for up to 7 consecutive nights] to determine the effects of the stress response on the stability of the intestinal microbiota. In this initial experiment, approximately 100,000 sequences from the cecal contents of 32 mice (approximately 3000 sequences per mouse) were identified to characterize the community structure of the cecal microbiota (Bailey et al., 2010). Overall bacterial community diversity and richness decreased with repeated exposure of prolonged restraint. In addition, hierarchical cluster analyses indicated that the profile of the top ten most abundant bacterial types was significantly different in the mice exposed to 3, 5, or 7 days of restraint compared to profiles in control mice or mice only exposed to 1 day of restraint. Interestingly, exposure to a single cycle of restraint resulted in a bacterial profile that was similar to profiles found in mice deprived of food and water during the same time that stressed-mice were exposed to the restraint stressor (Bailey et al., 2010). This indicates that some of the effects of the stressor on the microbiota are due to food and water deprivation, but upon repeated exposures, the stressor has unique effects on the microbiota that cannot be accounted for by changes in diet. In addition to affecting diversity and the relative abundances of individual types of microbiota, stressor exposure has also been shown to impact the total number of bacteria that can be cultured from the intestines (i.e., the total bacterial load). Chronic stressors, including the prolonged restraint stressor (Bailey et al., 2006) as well as 19 days of chronic subordinate colony housing (Reber et al., 2011) were sufficient to significantly increase the number of bacteria that could be cultured from the intestines. This is important, because an overgrowth of intestinal bacteria is a predisposing factor for the translocation of bacteria from the lumen of the intestines to the interior of the body (Berg, 1999). While translocation of microbes into systemic sites, such as the spleen or liver, can result in severe inflammatory conditions, translocation/penetration of microbes across the intestinal epithelial barrier into deeper layers of the intestinal tissue (e.g., into the lamina propria) can lead to intestinal inflammation. Microbiota-induced intestinal inflammation is thought to contribute to the pathophysiology of functional gastrointestinal disorders, such as irritable bowel syndrome (Collins et al., 2009), as well as inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis (Sartor, 2008, 2009). These diseases are often exacerbated during periods of stress (Drossman, 1998; Maunder and Levenstein, 2008;

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Mawdsley and Rampton, 2006; Rampton, 2009), which could be the consequence of stressor-induced increases in microbial load and penetration into intestinal tissue. This was nicely demonstrated by Reber et al. (2011), who demonstrated that chronic subordinate colony housing resulted in the development of spontaneous colitis. This colitis could be prevented by treating animals with broad spectrum antibiotics to reduce the bacterial load in the intestines, thus demonstrating that the microbiota are involved in the initiation of the colitis (Reber et al., 2011; also reviewed in Reber, 2012). However, the microbiota are not the sole players in stressor-induced increases in colonic inflammation. Stressor exposure is known to induce mucosal mast cell degranulation (Cameron and Perdue, 2005; Santos et al., 2001) (which facilitates the penetration of microbes into host tissue) (Soderholm et al., 2002) and increases the expression of pattern recognition receptors, like Toll-like receptors (TLR), within the intestinal tissue (McKernan et al., 2009). The TLR lead to the production of chemokines and inflammatory cytokines to recruit and activate immune cells to the affected intestine. Additional studies are needed, including studies involving germfree and reconstituted germfree mice, to determine whether stressor-induced alterations in mucosal immunity lead to alterations in the microbiota community structure, whether alterations in microbiota community structure lead to alterations in mucosal immunity, or whether the two are truly interdependent upon each other. Effects on gut microbiota function It is generally believed that reducing microbial diversity and richness in the intestines in turn enhances susceptibility to enteric pathogens. Thus, it was hypothesized that exposing mice to the prolonged restraint stressor prior to pathogen challenge would in turn increase susceptibility/the severity of infectious challenge (Bailey et al., 2010). To test this hypothesis, mice were orally challenged with Citrobacter rodentium. C. rodentium is a natural murine colonic pathogen, with pathogenesis and resulting colonic pathology that are nearly indistinguishable from that produced in humans infected with enteropathogenic Escherichia coli, and some components of enterrohemmorhagic E. coli (Borenshtein et al., 2008; Luperchio and Schauer, 2001; Mundy et al., 2005). As the infection progresses, the colonic inflammatory response resembles many aspects of the colitis found in patients with inflammatory bowel disease (Eckmann, 2006; Mundy et al., 2005). Exposing mice to the prolonged restraint stressor prior to C. rodentium challenge significantly increased pathogen colonization. This increased colonization was associated with an increased inflammatory response in the colon marked by increased inflammatory cytokine (e.g., TNF-α) mRNA levels and increased colonic histopathology (Bailey et al., 2010). It is not likely that the stressor-induced increase in C. rodentium colonization was due to immunosuppression (which can occur in response to prolonged restraint) since measures of mucosal immunity were either unaffected or even enhanced prior to pathogen challenge (Bailey et al., 2010). In addition, preliminary studies indicate that stressor-induced alterations of the microbiota can impact pathogen colonization, particularly within the first week after pathogen challenge (data not shown). Moreover, preliminary studies indicate that stressor-induced changes in the microbiota lead to increases in the colonic inflammatory response upon pathogen challenge (data not shown). One research goal of this laboratory is to clearly define the mechanisms by which the microbiota can impact stressor-induced increases in the severity of colonic infection and to determine whether/ how the microbiota regulate the mucosal inflammatory response. Studies involving the social disruption stressor Effects on gut microbiota community structure Prolonged or chronic stressors are not the only stressors that can impact the structure of the microbiota. In a separate study, a social

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stressor, called social disruption (SDR), was used to study the effects of the stress response on the microbiota (Bailey et al., 2011). Murine social stressors, including the SDR stressor, involve aggressive interactions between conspecifics, and are widely used to study the effects of stress on behavior and on physiological functioning (Bohus et al., 1993; de Groot et al., 1999; Korte et al., 1990; Sgoifo et al., 1998). The SDR stressor involves both physical and psychological components, and the defeated mice develop anxiety-like behaviors (Kinsey et al., 2007; Wohleb et al., 2011) and a physiological stress response marked by elevated corticosterone (Bailey et al., 2004; Engler et al., 2005), epinephrine, and norepinephrine (Hanke et al., 2006). Importantly, exposure to the SDR stressor has well defined effects on systemic immune responses. For example, exposure to the SDR stressor is known to increase circulating levels of cytokines, such as IL-1 and IL-6 (even in the absence of infectious challenge) (Avitsur et al., 2005; Engler et al., 2008; Stark et al., 2002), which is also commonly evident in humans exposed to different types of stressors (Brydon and Steptoe, 2005; Brydon et al., 2004; Steptoe et al., 2007). In addition, exposure to the SDR stressor reduces the sensitivity of splenic monocytes/macrophages to the suppressive effects of glucocorticoid hormones and increases the ability of these splenic monocytes/ macrophages to kill target microbes (Avitsur et al., 2001; Bailey et al., 2004, 2007; Engler et al., 2005; Stark et al., 2001). Thus, this welldefined stressor was used to assess the impact of a social stressor on microbial diversity in the cecum. As with prolonged restraint, exposure to the SDR stressor for 2 h per day on 6 consecutive days reduced microbial diversity and richness in the cecal contents. This was manifest 15 h after the last exposure to the stressor, but not immediately following the last cycle of the stressor (Bailey et al., 2011) suggesting that some of the effects of the stressor occur over longer periods of time. Interestingly, the relative abundance of 3 members of the microbiota (i.e., Coprococcus spp., Pseudobutyrivibrio spp., and Dorea spp.) were inversely correlated with stressor-induced increases in circulating IL-6 (Bailey et al., 2011). The nature of the association between these bacterial species and circulating IL-6 is not yet clear, but the finding of significant correlations between gut microbiota and circulating cytokines suggested that it was possible that microbial populations were directly influencing systemic immune responses. To determine whether there was a

causative relation between the microbiota and stressor-induced increases in circulating cytokines, the microbiota were reduced by orally administering an antibiotic cocktail prior to, as well as during exposure to the SDR stressor. Antibiotic administration significantly reduced the stressor-induced increase in circulating cytokines, such as IL-6, indicating that the microbiota were necessary for the effects of the stressor to manifest (Bailey et al., 2011). It is interesting to note that stressor-induced increases in circulating IL-6 have previously been linked to activation of the sympathetic nervous system (SNS) (Cole et al., 2010). Blocking the activation of the SNS through the use of α- and β-adrenergic antagonists was reported to block the stressor-induced increases in innate immunity (Hanke et al., 2008; Johnson et al., 2005). These previous studies do not eliminate a potential role for the microbiota. Stressor-induced activation of the SNS can significantly impact gut functioning, which may in turn impact the structure of the microbiota (Collins and Bercik, 2009; Lomax et al., 2010). In addition, SNS-derived catecholamines, primarily norepinephrine, have the capacity to stimulate the growth of many enteric bacteria (Freestone et al., 2000, 2002; Lyte and Nguyen, 1997). Thus, it is possible that stressor-induced SNS activity impacts circulating cytokines through direct effects on leukocytes and/or indirectly through effects on the microbiota. Effects on gut microbiota function Exposure to the SDR stressor increases the capacity of splenic macrophages to kill target microbes (Bailey et al., 2007). And, the ability of phagocytes to kill target microbes has previously been linked to the intestinal microbiota. Phagocytes from mice lacking microbiota were deficient in their ability to kill target pathogens, i.e., Streptococcus pneumoniae and Staphylococcus aureus (Clarke et al., 2010). This led to the hypothesis that stressor-induced increases in the ability of splenic macrophages to kill a target microbe, i.e., E. coli, would also be affected by the intestinal microbiota. To test this hypothesis, mice were treated with broad spectrum antibiotics to reduce the microbiota. As predicted, treating mice with antibiotics reduced the stressor-induced increase in splenic macrophage microbicidal activity (Allen et al., 2012). This effect was associated with a significant decrease in macrophage peroxynitrite production, which is necessary for the enhancive effect of the stressor on

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Fig. 1. Inhibiting mast cell degranulation impacts the SDR stressor-induced increase in splenic macrophage activity. Mice were exposed to the SDR stressor for 2 h at the beginning of the active cycle on 6 consecutive days as previously described (Allen et al., 2012; Bailey et al., 2011). Mice were treated with vehicle or with cromolyn sodium salt (50 mg/kg intraperitoneally) 1 h prior to each cycle of SDR to inhibit mast cell degranulation. The morning following the 6th cycle of SDR, the mice were euthanized and splenic macrophages isolated. A.). To assess microbicidal activity, the splenic macrophages were co-cultured with Escherichia coli (ATCC 10798) in duplicate. After 20 min of co-culture, the number of bacteria contained within the macrophages was counted using agar plates. In the duplicate wells, extracellular bacteria were washed away and the macrophages cultured for an additional 70 min. At the end of the 90 min total period, the number of bacteria remaining alive was determined via plating. Exposing vehicle-treated mice to the SDR stressor increased the microbicidal activity of splenic macrophages, as indicated by significantly reduced numbers of E. coli remaining alive within the macrophages at the 90 min period (F(1, 15) = 20.91, p b 0.001). However, inhibiting mast cell degranulation by treatment with cromolyn prevented the SDR stressor-induced effect (i.e., lack of an effect on bacterial killing (F(1, 15) = 0.73, not significant). B.) To determine the impact of the SDR stressor on the production of peroxynitrite, splenic macrophages were stimulated with phorbol myristate (PMA, 5 ng/ml), IFN-γ (5 ng/ml), and E. coli-derived LPS (1 μg/ml). Peroxynitrite was detected using a fluorescent dye (25 μM of 123-dihydrorhodamine) every 15 min using excitation and emission wavelengths of 544 nm and 618 nm. Peroxynitrite levels were higher in mice exposed to the SDR stressor (F(6, 120) = 17.76, p b 0.001). Administration of cromolyn to inhibit mast cell degranulation reduced peroxynitrite levels, as indicated by significantly higher levels of peroxynitrite in the vehicle-treated, SDR stressor mice compared to all other groups at 15, 30, 45, 60, and 75 min of stimulation (*p b 0.05 vs. all other groups at each time point). n = 6–12 per group.

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A) TNF - α mRNA Mast Cell Inhibitor

Vehicle 30

Unstimulated LPS-Stimulated

Fold Increase Over HCC Control (Unstimulated)

Fold Increase Over HCC Control (Unstimulated)

30

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25 20 15 10 5 0 HCC Control

Unstimulated LPS-Stimulated

25 20 15 10 5 0

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B) IL-1β mRNA Vehicle

Mast Cell Inhibitor 12

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Fold Increase Over HCC Control (Unstimulated)

12

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8 6 4 2 0 HCC Control

Unstimulated LPS-Stimulated

10 8 6 4 2 0

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SDR Stressor

Fig. 2. Inhibiting mast cell degranulation in turn reduces the SDR stressor-induced increase in inflammatory cytokine mRNA in LPS-stimulated splenic macrophages. Mice were exposed to the SDR stressor for 2 h at the beginning of the active cycle on 6 consecutive days as previously described (Allen et al., 2012; Bailey et al., 2012). Mice were treated with vehicle or with cromolyn sodium salt (50 mg/kg intraperitoneally) 1 h prior to each cycle of SDR to inhibit mast cell degranulation. The morning following the 6th cycle of SDR, the mice were euthanized and splenic macrophages isolated. These cells were stimulated for 90 min with 1 μg/ml of E. coli-derived LPS. After the 90 min stimulation, cytokine mRNA was assessed using real-time PCR as described previously (Allen et al., 2012). A.) Stimulating splenic macrophages from SDR stressor-exposed mice that were treated with vehicle resulted in a significant increase in TNF-α mRNA (F(1, 13) = 13.05, p b 0.05). Treating mice with cromolyn to prevent mast cell degranulation prevented this stressor-induced increase in TNF-α mRNA levels (F(1, 13) = 0.02, not significant. B.) Stimulating splenic macrophages from vehicle-treated, SDR stressor-exposed mice also increased IL-1β mRNA (F(1, 15) = 11.50, p b 0.05). Again, treating mice with cromolyn to inhibit mast cell degranulation prevented this stressor-induced increase in IL-1β mRNA (F(1, 15) = 0.12, not significant). n = 4–5 per group.

microbicidal activity (unpublished observation). To be sure the effects were due to a lack of microbiota, and not an indirect side effect of the antibiotics, germfree mice that have never come into contact

with microbes were also exposed to the SDR stressor. Exposing germfree mice to the stressor did not enhance the activity of splenic macrophages. However, colonizing the germfree mice with microbiota

Disruption of Homeostatic Interactions Between Host and Microbiota

Stressor-Induced Physiological Response e.g., Glucocorticoids, Catecholamines

Mucosal Effects Systemic Effects Inflammatory Cytokines - e.g., IL-1

Mucosal Mast Cell Degranulation

Susceptibility to Mucosal Pathogens -Mechanism of Action -Disrupted barrier exclusion? - Intestinal Inflammation? -Dysregulated immune activity? Inflammation

Translocation of Live Microbiota or Bacterial Peptidoglycan

Enhanced Microbicidal Activity - Peroxynitrite - Superoxide - Nitric Oxide Enhanced Cytokine Production

Fig. 3. Proposed mechanism linking intestinal microbiota to stressor-induced alterations in mucosal and systemic immune responses. Stressor exposure activates an endocrine response that can disrupt homeostatic interactions between the microbiota and the mucosal immune system. Disrupting these homeostatic interactions can increase susceptibility to enteric pathogens through as yet undefined mechanisms. Disrupted homeostatic interactions are also hypothesized to result in altered chemokine and cytokine responses by intestinal epithelial cells. Systemic immune responses can also be affected when mast cell degranulation increases the permeability of the intestinal barrier by disrupting tight junction proteins found between intestinal epithelial cells. It is hypothesized that this enhanced barrier permeability increases the translocation of living microbes and/or bacterial products from the lumen of the intestines to the systemic circulation where they prime splenic monocytes/macrophages for enhanced reactivity. It is hypothesized that this priming is the result of either direct effects of microbial products, or the effects of other microbiota-associated factors.

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prior to exposure to the SDR stressor again led to significant increases in splenic macrophage microbicidal activity (Allen et al., 2012). These data demonstrate that the microbiota are involved in stressorinduced increases in splenic macrophage reactivity to microbial stimuli. Importance of mast cells in linking the microbiota to stressor-induced immunomodulation The mechanisms linking the microbiota to stressor-induced alterations of splenic macrophage activity are not yet clearly understood, but could involve the translocation of bacteria and/or bacterial products from the lumen of the intestines to the interior of the body. Previous studies have shown that exposure to the SDR stressor increases the prevalence of living bacteria within regional lymph nodes (Bailey et al., 2006) and also increases the levels of bacterial-derived peptidoglycan found in the circulation of uninfected animals (Allen et al., 2012). These findings may seem somewhat surprising, because the well-developed tight junctions between intestinal epithelial cells provide a nearly impervious barrier to microbes, microbial antigens, or other soluble molecules. However, several different types of stressors have been shown to increase the permeability of this intestinal barrier through mast cell-dependent mechanisms (Cameron and Perdue, 2005; Santos et al., 2001; Soderholm et al., 2002). These previous studies led to the hypothesis that intestinal mast cells are an important link between the intestinal microbiota and stressor-induced increases in splenic macrophage reactivity to microbial stimuli. To test this hypothesis, mice were treated with a mast cell stabilizer (i.e., cromolyn) prior to, as well as during exposure to the SDR stressor. Preventing mast cell degranulation in turn prevented the stressor-induced increase in macrophage microbicidal activity (Fig. 1A). This effect was associated with a significant reduction in splenic macrophage production of peroxynitrite (Fig. 1B) which is necessary for the stressor-induced increase in splenic macrophage microbicidal activity (unpublished observations). Inhibiting mast cell degranulation also reduced the effects of the stressor on inflammatory cytokine production. Splenic macrophages from mice exposed to the SDR stressor had higher gene expression for TNF-α as well as IL-1β after LPS stimulation. Inhibiting mast cell degranulation in turn reduced the stressor-induced increases in both TNF-α and IL1β (Fig. 2). These data demonstrate that both the microbiota and mast cells contribute to stressor-induced increases in splenic macrophage reactivity to microbial stimuli. Current studies are determining whether stressor-induced mast cell degranulation leads to a leaky intestinal barrier that is permissive to the translocation of bacteria and/ or bacterial products from the lumen of the intestines to the interior of the body. Studies are also assessing whether the translocated microbes directly prime cells of the innate immune system, or whether mediating factors such as heat shock proteins (Fleshner et al., 2010) or inflammatory cytokines like IL-1β (unpublished observations) contribute to the effects on splenic macrophages. Conclusions It has long been recognized that the body is colonized by microbes and that these microbes have beneficial health effects. However, the development of culture-independent deep sequencing methodologies has revealed the amazing density and diversity of commensal microbes. As interest in the microbiota has increased, so too has the awareness of the wide range of effects that microbes have on the host. Many of the functions of the microbiota are related to community structure. These structure–function relationships have developed through the co-evolution of the host and its microbiota, such that alterations in one typically lead to alterations in the other. This phenomenon is clearly evident in the complex interactions between the microbiota and the immune system, which can be significantly

altered during a physiological stress response as illustrated in Fig. 3. During periods of quiescence, homeostatic interactions occur between the host and its microbiota that maintain beneficial microbial populations and limit the induction of mucosal inflammatory responses (Fig. 3). The studies presented in this review indicate that exposing laboratory animals, as well as humans, to environmental or psychological stressors significantly disrupts these homeostatic interactions. These disruptions are manifest as alterations in the community structure of the intestinal microbiota and increased host inflammatory responses. Within the model shown in Fig. 3, it is interesting to postulate the evolutionary context within which stressor-induced alterations of the microbiota and host inflammatory responses developed. It is possible that the stressor-induced microbial alterations are reflective of gastrointestinal physiological responses that are meant to be protective against enteric pathogens. Exposing experimental animals to stressful stimuli increases contractions and secretory activity in the colon (Fone et al., 1990; Saunders et al., 2002; Tache et al., 1999; Wood, 2007). Many enteric pathogens are non-invasive and cause disease by colonizing the lumenal surface of the colonic gastrointestinal epithelium. Thus, increased colonic secretions and motility can be beneficial because they help to “flush” existing microbes from the colon. If commensal microbes that reduce colonic inflammatory responses, such as L. reuteri, are also reduced due to the colonic physiological response to stress, the reduction would result in an internal environment that is conducive to the development of a mucosal inflammatory response. While this may be adaptive in the face of challenge with some types of pathogens, other pathogens including C. rodentium and Salmonella enterica subspecies Typhimurium, more efficiently colonize the inflamed colon (Lupp et al., 2007; Stecher et al., 2007). The mechanisms by which this enhanced colonization occurs are not yet completely understood, but it is possible that they reflect evolutionary pressures that have selected for pathogens able to colonize in the face of inflammatory reactions. Infections that are limited to mucosal surfaces tend to be self limiting. If, however, these enteric pathogens enter into the systemic circulation, they can cause severe disease. Thus, it may still be beneficial for stressor-induced alterations in the microbiota to lead to enhanced immune responses if the enhancement can control or prevent the systemic spread of the pathogen. Stressor exposure is known to increase innate immune responses (Brydon et al., 2005; Campisi et al., 2003; Dhabhar, 2009), but understanding the mechanisms by which this occurs has been difficult. The data presented in this review indicate that the microbiota play an essential role in stressor-induced immune enhancement. During periods of quiescence, the immune system is largely ignorant of the microbiota due to the selectively permeable intestinal barrier. During stressor exposure, however, nervous system-induced mast cell degranulation can significantly increase intestinal permeability (Soderholm and Perdue, 2001). It is proposed that this increased permeability leads to the translocation of intact microbiota and/or their products or components such as peptidoglycan (as outlined in Fig. 3). It is further hypothesized that this translocation facilitates stressor-induced increases in circulating cytokines, such as IL-1α/β and IL-6 even in the absence of pathogen challenge. It is hypothesized that these cytokines, or other microbiotaassociated mediating factors like heat shock proteins, prime splenic monocytes/macrophages for enhanced microbicidal activity. This stressor-induced enhancement is beneficial to the host when pathogens are not confined to the intestines and enter into the systemic circulation. When considered together, it is evident that microbiota are interactively involved in stressor-induced immunomodulation at the mucosal surface, as well as at systemic sites. As interest in the microbiota continues to build, it will be of importance to identify both adaptive and maladaptive consequences of the physiological stress response on host–microbe interactions.

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Acknowledgments I gratefully acknowledge the helpful contributions of Dr. Rebecca G. Allen, Mr. Jeffrey Galley, and Ms. Amy Hufnagle. I would also like to acknowledge the helpful discussion with Dr. Mark Lyte. References Allen, R.G., Lafuse, W.P., Galley, J.D., Ali, M.M., Ahmer, B.M., Bailey, M.T., 2012. The intestinal microbiota are necessary for stressor-induced enhancement of splenic macrophage microbicidal activity. Brain Behav. Immun. 26, 371–382. Allison, S.D., Martiny, J.B., 2008. Colloquium paper: resistance, resilience, and redundancy in microbial communities. Proc. Natl. Acad. Sci. U. S. A. 105 (Suppl. 1), 11512–11519. Antonopoulos, D.A., Huse, S.M., Morrison, H.G., Schmidt, T.M., Sogin, M.L., Young, V.B., 2009. Reproducible community dynamics of the gastrointestinal microbiota following antibiotic perturbation. Infect. Immun. 77, 2367–2375. Artis, D., 2008. Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut. Nat. Rev. Immunol. 8, 411–420. Avitsur, R., Stark, J.L., Sheridan, J.F., 2001. Social stress induces glucocorticoid resistance in subordinate animals. Horm. Behav. 39, 247–257. Avitsur, R., Kavelaars, A., Heijnen, C., Sheridan, J.F., 2005. Social stress and the regulation of tumor necrosis factor-alpha secretion. Brain Behav. Immun. 19, 311–317. Bailey, M.T., Coe, C.L., 1999. Maternal separation disrupts the integrity of the intestinal microflora in infant rhesus monkeys. Dev. Psychobiol. 35, 146–155. Bailey, M.T., Avitsur, R., Engler, H., Padgett, D.A., Sheridan, J.F., 2004. Physical defeat reduces the sensitivity of murine splenocytes to the suppressive effects of corticosterone. Brain Behav. Immun. 18, 416–424. Bailey, M.T., Engler, H., Sheridan, J.F., 2006. Stress induces the translocation of cutaneous and gastrointestinal microflora to secondary lymphoid organs of C57BL/6 mice. J. Neuroimmunol. 171, 29–37. Bailey, M.T., Engler, H., Powell, N.D., Padgett, D.A., Sheridan, J.F., 2007. Repeated social defeat increases the bactericidal activity of splenic macrophages through a Tolllike receptor-dependent pathway. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1180–R1190. Bailey, M.T., Dowd, S.E., Parry, N.M., Galley, J.D., Schauer, D.B., Lyte, M., 2010. Stressor exposure disrupts commensal microbial populations in the intestines and leads to increased colonization by Citrobacter rodentium. Infect. Immun. 78, 1509–1519. Bailey, M.T., Dowd, S.E., Galley, J.D., Hufnagle, A.R., Allen, R.G., Lyte, M., 2011. Exposure to a social stressor alters the structure of the intestinal microbiota: implications for stressor-induced immunomodulation. Brain Behav. Immun. 25, 397–407. Berg, R.D., 1999. Bacterial translocation from the gastrointestinal tract. Adv. Exp. Med. Biol. 473, 11–30. Bernet-Camard, M.F., Lievin, V., Brassart, D., Neeser, J.R., Servin, A.L., Hudault, S., 1997. The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo. Appl. Environ. Microbiol. 63, 2747–2753. Bohus, B., Koolhaas, J.M., Heijnen, C.J., de Boer, O., 1993. Immunological responses to social stress: dependence on social environment and coping abilities. Neuropsychobiology 28, 95–99. Borenshtein, D., McBee, M.E., Schauer, D.B., 2008. Utility of the Citrobacter rodentium infection model in laboratory mice. Curr. Opin. Gastroenterol. 24, 32–37. Brydon, L., Steptoe, A., 2005. Stress-induced increases in interleukin-6 and fibrinogen predict ambulatory blood pressure at 3-year follow-up. J. Hypertens. 23, 1001–1007. Brydon, L., Edwards, S., Mohamed-Ali, V., Steptoe, A., 2004. Socioeconomic status and stress-induced increases in interleukin-6. Brain Behav. Immun. 18, 281–290. Brydon, L., Edwards, S., Jia, H., Mohamed-Ali, V., Zachary, I., Martin, J.F., Steptoe, A., 2005. Psychological stress activates interleukin-1beta gene expression in human mononuclear cells. Brain Behav. Immun. 19, 540–546. Buynitsky, T., Mostofsky, D.I., 2009. Restraint stress in biobehavioral research: recent developments. Neurosci. Biobehav. Rev. 33, 1089–1098. Cameron, H.L., Perdue, M.H., 2005. Stress impairs murine intestinal barrier function: improvement by glucagon-like peptide-2. J. Pharmacol. Exp. Ther. 314, 214–220. Campisi, J., Leem, T.H., Fleshner, M., 2003. Stress-induced extracellular Hsp72 is a functionally significant danger signal to the immune system. Cell Stress Chaperones 8, 272–286. Clarke, T.B., Davis, K.M., Lysenko, E.S., Zhou, A.Y., Yu, Y., Weiser, J.N., 2010. Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity. Nat. Med. 16, 228–231. Coconnier, M.H., Bernet, M.F., Chauviere, G., Servin, A.L., 1993a. Adhering heat-killed human Lactobacillus acidophilus, strain LB, inhibits the process of pathogenicity of diarrhoeagenic bacteria in cultured human intestinal cells. J. Diarrhoeal Dis. Res. 11, 235–242. Coconnier, M.H., Bernet, M.F., Kerneis, S., Chauviere, G., Fourniat, J., Servin, A.L., 1993b. Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiol. Lett. 110, 299–305. Cole, S.W., Arevalo, J.M., Takahashi, R., Sloan, E.K., Lutgendorf, S.K., Sood, A.K., Sheridan, J.F., Seeman, T.E., 2010. Computational identification of gene-social environment interaction at the human IL6 locus. Proc. Natl. Acad. Sci. U. S. A. 107, 5681–5686. Collins, S.M., Bercik, P., 2009. The relationship between intestinal microbiota and the central nervous system in normal gastrointestinal function and disease. Gastroenterology 136, 2003–2014.

293

Collins, S.M., Denou, E., Verdu, E.F., Bercik, P., 2009. The putative role of the intestinal microbiota in the irritable bowel syndrome. Dig. Liver Dis. 41, 850–853. de Groot, J., van Milligen, F.J., Moonen-Leusen, B.W., Thomas, G., Koolhaas, J.M., 1999. A single social defeat transiently suppresses the anti-viral immune response in mice. J. Neuroimmunol. 95, 143–151. Dethlefsen, L., Huse, S., Sogin, M.L., Relman, D.A., 2008. The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing. PLoS Biol. 6, e280. Dhabhar, F.S., 2009. Enhancing versus suppressive effects of stress on immune function: implications for immunoprotection and immunopathology. Neuroimmunomodulation 16, 300–317. Dobbs, C.M., Vasquez, M., Glaser, R., Sheridan, J.F., 1993. Mechanisms of stress-induced modulation of viral pathogenesis and immunity. J. Neuroimmunol. 48, 151–160. Dobbs, C.M., Feng, N., Beck, F.M., Sheridan, J.F., 1996. Neuroendocrine regulation of cytokine production during experimental influenza viral infection: effects of restraint stress-induced elevation in endogenous corticosterone. J. Immunol. 157, 1870–1877. Drossman, D.A., 1998. Presidential address: gastrointestinal illness and the biopsychosocial model. Psychosom. Med. 60, 258–267. Eaton, K.A., Honkala, A., Auchtung, T.A., Britton, R.A., 2011. Probiotic Lactobacillus reuteri ameliorates disease due to enterohemorrhagic Escherichia coli in germfree mice. Infect. Immun. 79, 185–191. Eckburg, P.B., Bik, E.M., Bernstein, C.N., Purdom, E., Dethlefsen, L., Sargent, M., Gill, S.R., Nelson, K.E., Relman, D.A., 2005. Diversity of the human intestinal microbial flora. Science 308, 1635–1638. Eckmann, L., 2006. Animal models of inflammatory bowel disease: lessons from enteric infections. Ann. N. Y. Acad. Sci. 1072, 28–38. Engler, H., Engler, A., Bailey, M.T., Sheridan, J.F., 2005. Tissue-specific alterations in the glucocorticoid sensitivity of immune cells following repeated social defeat in mice. J. Neuroimmunol. 163, 110–119. Engler, H., Bailey, M.T., Engler, A., Stiner-Jones, L.M., Quan, N., Sheridan, J.F., 2008. Interleukin-1 receptor type 1-deficient mice fail to develop social stress-associated glucocorticoid resistance in the spleen. Psychoneuroendocrinology 33, 108–117. Everson, C.A., Toth, L.A., 2000. Systemic bacterial invasion induced by sleep deprivation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278, R905–R916. Fleshner, M., Maslanik, T., Beninson, L., 2010. In vivo tissue source and releasing signal of endogenous extracellular Hsp72. In: Asea, A.A.A., Pederson, B.K. (Eds.), Heat Shock Proteins and Whole Body Physiology. Springer Publishing Group, New York, pp. 193–215. Fone, D.R., Horowitz, M., Maddox, A., Akkermans, L.M., Read, N.W., Dent, J., 1990. Gastroduodenal motility during the delayed gastric emptying induced by cold stress. Gastroenterology 98, 1155–1161. Food and Agriculture Organization of the United Nations & World Health Organization, 2001. Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria. In. Frank, D.N., St Amand, A.L., Feldman, R.A., Boedeker, E.C., Harpaz, N., Pace, N.R., 2007. Molecular–phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc. Natl. Acad. Sci. U. S. A. 104, 13780–13785. Frank, D.N., Robertson, C.E., Hamm, C.M., Kpadeh, Z., Zhang, T., Chen, H., Zhu, W., Sartor, R.B., Boedeker, E.C., Harpaz, N., Pace, N.R., Li, E., 2011. Disease phenotype and genotype are associated with shifts in intestinal-associated microbiota in inflammatory bowel diseases. Inflamm. Bowel Dis. 17, 179–184. Freestone, P.P., Lyte, M., Neal, C.P., Maggs, A.F., Haigh, R.D., Williams, P.H., 2000. The mammalian neuroendocrine hormone norepinephrine supplies iron for bacterial growth in the presence of transferrin or lactoferrin. J. Bacteriol. 182, 6091–6098. Freestone, P.P., Williams, P.H., Haigh, R.D., Maggs, A.F., Neal, C.P., Lyte, M., 2002. Growth stimulation of intestinal commensal Escherichia coli by catecholamines: a possible contributory factor in trauma-induced sepsis. Shock 18, 465–470. Frese, S.A., Benson, A.K., Tannock, G.W., Loach, D.M., Kim, J., Zhang, M., Oh, P.L., Heng, N.C., Patil, P.B., Juge, N., Mackenzie, D.A., Pearson, B.M., Lapidus, A., Dalin, E., Tice, H., Goltsman, E., Land, M., Hauser, L., Ivanova, N., Kyrpides, N.C., Walter, J., 2011. The evolution of host specialization in the vertebrate gut symbiont Lactobacillus reuteri. PLoS Genet. 7, e1001314. Fukuda, S., Toh, H., Hase, K., Oshima, K., Nakanishi, Y., Yoshimura, K., Tobe, T., Clarke, J.M., Topping, D.L., Suzuki, T., Taylor, T.D., Itoh, K., Kikuchi, J., Morita, H., Hattori, M., Ohno, H., 2011. Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature 469, 543–547. Hanke, M.L., Bailey, M.T., Sheridan, J.F., 2006. Repeated Social Defeat Increases Catecholamine Production in the Spleen, Liver Mediastinal Lymph Nodes, and Brain of CD-1 Mice. Brain Behav. Immun. 20 (3 Supplement), 30. Hanke, M.L., Bailey, M.T., Powell, N.D., Stiner, L., Sheridan, J.F., 2008. Beta-2 adrenergic blockade decreases the immunomodulatory effects of Social Disruption Stress (SDR). Brain Behav. Immun. 22 (4 Supplement), 39–40. Holdeman, L.V., Good, I.J., Moore, W.E., 1976. Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Appl. Environ. Microbiol. 31, 359–375. Huffnagle, G.B., 2010. The microbiota and allergies/asthma. PLoS Pathog. 6, e1000549. Johnson, J.D., Campisi, J., Sharkey, C.M., Kennedy, S.L., Nickerson, M., Greenwood, B.N., Fleshner, M., 2005. Catecholamines mediate stress-induced increases in peripheral and central inflammatory cytokines. Neuroscience 135, 1295–1307. Jones, S.E., Versalovic, J., 2009. Probiotic Lactobacillus reuteri biofilms produce antimicrobial and anti-inflammatory factors. BMC Microbiol. 9, 35. Kaser, A., Zeissig, S., Blumberg, R.S., 2010. Inflammatory bowel disease. Annu. Rev. Immunol. 28, 573–621. Kinsey, S.G., Bailey, M.T., Sheridan, J.F., Padgett, D.A., Avitsur, R., 2007. Repeated social defeat causes increased anxiety-like behavior and alters splenocyte function in C57BL/6 and CD-1 mice. Brain Behav. Immun. 21, 458–466.

294

M.T. Bailey / Hormones and Behavior 62 (2012) 286–294

Knowles, S.R., Nelson, E.A., Palombo, E.A., 2008. Investigating the role of perceived stress on bacterial flora activity and salivary cortisol secretion: a possible mechanism underlying susceptibility to illness. Biol. Psychol. 77, 132–137. Korte, S.M., Smit, J., Bouws, G.A.H., Koolhaas, J.M., Bohus, B., 1990. Behavioral and neuroendocrine response to psychosocial stress in male rats: the effects of the 5-HT1A agonistic ipsapirone. Horm. Behav. 24, 554–567. Ley, R.E., Backhed, F., Turnbaugh, P., Lozupone, C.A., Knight, R.D., Gordon, J.I., 2005. Obesity alters gut microbial ecology. Proc. Natl. Acad. Sci. U. S. A. 102, 11070–11075. Li, Q., Verma, I.M., 2002. NF-kappaB regulation in the immune system. Nat. Rev. Immunol. 2, 725–734. Lizko, N.N., 1987. Stress and intestinal microflora. Nahrung 31, 443–447. Lomax, A.E., Sharkey, K.A., Furness, J.B., 2010. The participation of the sympathetic innervation of the gastrointestinal tract in disease states. Neurogastroenterol. Motil. 22, 7–18. Luperchio, S.A., Schauer, D.B., 2001. Molecular pathogenesis of Citrobacter rodentium and transmissible murine colonic hyperplasia. Microbes Infect. 3, 333–340. Lupp, C., Robertson, M.L., Wickham, M.E., Sekirov, I., Champion, O.L., Gaynor, E.C., Finlay, B.B., 2007. Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae. Cell Host Microbe 2, 119–129. Lyte, M., Nguyen, K.T., 1997. Alteration of Escherichia coli O157:H7 growth and molecular fingerprint by the neuroendocrine hormone noradrenaline. Microbios 89, 197–213. Makino, S., Ikegami, S., Kano, H., Sashihara, T., Sugano, H., Horiuchi, H., Saito, T., Oda, M., 2006. Immunomodulatory effects of polysaccharides produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1. J. Dairy Sci. 89, 2873–2881. Marchiando, A.M., Graham, W.V., Turner, J.R., 2010. Epithelial barriers in homeostasis and disease. Annu. Rev. Pathol. 5, 119–144. Maunder, R.G., Levenstein, S., 2008. The role of stress in the development and clinical course of inflammatory bowel disease: epidemiological evidence. Curr. Mol. Med. 8, 247–252. Mawdsley, J.E., Rampton, D.S., 2006. The role of psychological stress in inflammatory bowel disease. Neuroimmunomodulation 13, 327–336. McKernan, D.P., Nolan, A., Brint, E.K., O'Mahony, S.M., Hyland, N.P., Cryan, J.F., Dinan, T.G., 2009. Toll-like receptor mRNA expression is selectively increased in the colonic mucosa of two animal models relevant to irritable bowel syndrome. PLoS One 4, e8226. Metchnikoff, E., 1908. The Prolongation of Life. GP Putnam's Sons, New York. Mundy, R., MacDonald, T.T., Dougan, G., Frankel, G., Wiles, S., 2005. Citrobacter rodentium of mice and man. Cell. Microbiol. 7, 1697–1706. Neish, A.S., 2004. Molecular aspects of intestinal epithelial cell-bacterial interactions that determine the development of intestinal inflammation. Inflamm. Bowel Dis. 10, 159–168. Nenci, A., Becker, C., Wullaert, A., Gareus, R., van, L.G., Danese, S., Huth, M., Nikolaev, A., Neufert, C., Madison, B., Gumucio, D., Neurath, M.F., Pasparakis, M., 2007. Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature 446, 557–561. O'Hara, A.M., Shanahan, F., 2006. The gut flora as a forgotten organ. EMBO Rep. 7, 688–693. Ott, S.J., Musfeldt, M., Wenderoth, D.F., Hampe, J., Brant, O., Folsch, U.R., Timmis, K.N., Schreiber, S., 2004. Reduction in diversity of the colonic mucosa associated bacterial microflora in patients with active inflammatory bowel disease. Gut 53, 685–693. Padgett, D.A., Sheridan, J.F., Dorne, J., Berntson, G.G., Candelora, J., Glaser, R., 1998. Social stress and the reactivation of latent herpes simplex virus type 1. Proc. Natl. Acad. Sci. U. S. A. 95, 7231–7235. Rampton, D., 2009. Does stress influence inflammatory bowel disease? The clinical data. Dig. Dis. 27 (Suppl. 1), 76–79. Reber, S.O., 2012. Stress and animal models of inflammatory bowel disease—an update on the role of the hypothalamo–pituitary–adrenal axis. Psychoneuroendocrinology 37, 1–19. Reber, S.O., Peters, S., Slattery, D.A., Hofmann, C., Scholmerich, J., Neumann, I.D., Obermeier, F., 2011. Mucosal immunosuppression and epithelial barrier defects are key events in murine psychosocial stress-induced colitis. Brain Behav. Immun. 25, 1153–1161. Rescigno, M., 2011. The intestinal epithelial barrier in the control of homeostasis and immunity. Trends Immunol. 32, 256–264. Resta, S.C., 2009. Effects of probiotics and commensals on intestinal epithelial physiology: implications for nutrient handling. J. Physiol. 587, 4169–4174. Robinson, C.J., Bohannan, B.J., Young, V.B., 2010. From structure to function: the ecology of host-associated microbial communities. Microbiol. Mol. Biol. Rev. 74, 453–476. Santos, J., Yang, P.C., Soderholm, J.D., Benjamin, M., Perdue, M.H., 2001. Role of mast cells in chronic stress induced colonic epithelial barrier dysfunction in the rat. Gut 48, 630–636. Sartor, R.B., 2008. Microbial influences in inflammatory bowel diseases. Gastroenterology 134, 577–594. Sartor, R.B., 2009. Microbial-host interactions in inflammatory bowel diseases and experimental colitis. Nestle Nutr. Workshop Ser. Pediatr. Program. 64, 121–132.

Saunders, P.R., Maillot, C., Million, M., Tache, Y., 2002. Peripheral corticotropinreleasing factor induces diarrhea in rats: role of CRF1 receptor in fecal watery excretion. Eur. J. Pharmacol. 435, 231–235. Sekirov, I., Russell, S.L., Antunes, L.C., Finlay, B.B., 2010. Gut microbiota in health and disease. Physiol. Rev. 90, 859–904. Sgoifo, A., Stilli, D., de Boer, S.F., Koolhaas, J.M., Musso, E., 1998. Acute social stress and cardiac electrical activity in rats. Aggress. Behav. 24, 287–296. Shibolet, O., Podolsky, D.K., 2007. TLRs in the Gut. IV. Negative regulation of Toll-like receptors and intestinal homeostasis: addition by subtraction. Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1469–G1473. Soderholm, J.D., Perdue, M.H., 2001. Stress and gastrointestinal tract. II. Stress and intestinal barrier function. Am. J. Physiol. Gastrointest. Liver Physiol. 280, G7–G13. Soderholm, J.D., Yang, P.C., Ceponis, P., Vohra, A., Riddell, R., Sherman, P.M., Perdue, M.H., 2002. Chronic stress induces mast cell-dependent bacterial adherence and initiates mucosal inflammation in rat intestine. Gastroenterology 123, 1099–1108. Stark, J.L., Avitsur, R., Padgett, D.A., Campbell, K.A., Beck, F.M., Sheridan, J.F., 2001. Social stress induces glucocorticoid resistance in macrophages. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, R1799–R1805. Stark, J.L., Avitsur, R., Hunzeker, J., Padgett, D.A., Sheridan, J.F., 2002. Interleukin-6 and the development of social disruption-induced glucocorticoid resistance. J. Neuroimmunol. 124, 9–15. Stecher, B., Robbiani, R., Walker, A.W., Westendorf, A.M., Barthel, M., Kremer, M., Chaffron, S., Macpherson, A.J., Buer, J., Parkhill, J., Dougan, G., von, M.C., Hardt, W.D., 2007. Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota. PLoS Biol. 5, 2177–2189. Steptoe, A., Hamer, M., Chida, Y., 2007. The effects of acute psychological stress on circulating inflammatory factors in humans: a review and meta-analysis. Brain Behav. Immun. 21, 901–912. Tache, Y., Martinez, V., Million, M., Rivier, J., 1999. Corticotropin-releasing factor and the brain–gut motor response to stress. Can. J. Gastroenterol. 13 (Suppl. A), 18A–25A. Tannock, G.W., Savage, D.C., 1974. Influences of dietary and environmental stress on microbial populations in the murine gastrointestinal tract. Infect. Immun. 9, 591–598. Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., Gordon, J.I., 2006. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031. Turnbaugh, P.J., Backhed, F., Fulton, L., Gordon, J.I., 2008. Diet-induced obesity is linked to marked but reversible alterations in the mouse distal gut microbiome. Cell Host Microbe 3, 213–223. Turnbaugh, P.J., Hamady, M., Yatsunenko, T., Cantarel, B.L., Duncan, A., Ley, R.E., Sogin, M.L., Jones, W.J., Roe, B.A., Affourtit, J.P., Egholm, M., Henrissat, B., Heath, A.C., Knight, R., Gordon, J.I., 2009. A core gut microbiome in obese and lean twins. Nature 457, 480–484. Walker, A.W., Sanderson, J.D., Churcher, C., Parkes, G.C., Hudspith, B.N., Rayment, N., Brostoff, J., Parkhill, J., Dougan, G., Petrovska, L., 2011. High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease. BMC Microbiol. 11, 7. Wikoff, W.R., Anfora, A.T., Liu, J., Schultz, P.G., Lesley, S.A., Peters, E.C., Siuzdak, G., 2009. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proc. Natl. Acad. Sci. U. S. A. 106, 3698–3703. Wohleb, E.S., Hanke, M.L., Corona, A.W., Powell, N.D., Stiner, L.M., Bailey, M.T., Nelson, R.J., Godbout, J.P., Sheridan, J.F., 2011. Beta-adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat. J. Neurosci. 31, 6277–6288. Wood, J.D., 2007. Effects of bacteria on the enteric nervous system: implications for the irritable bowel syndrome. J. Clin. Gastroenterol. 41 (Suppl. 1), S7–S19. Wu, G.D., Chen, J., Hoffmann, C., Bittinger, K., Chen, Y.Y., Keilbaugh, S.A., Bewtra, M., Knights, D., Walters, W.A., Knight, R., Sinha, R., Gilroy, E., Gupta, K., Baldassano, R., Nessel, L., Li, H., Bushman, F.D., Lewis, J.D., 2011. Linking long-term dietary patterns with gut microbial enterotypes. Science 334, 105–108. Xu, J., Gordon, J.I., 2003. Honor thy symbionts. Proc. Natl. Acad. Sci. U. S. A. 100, 10452–10459. Yasuda, E., Serata, M., Sako, T., 2008. Suppressive effect on activation of macrophages by Lactobacillus casei strain Shirota genes determining the synthesis of cell wallassociated polysaccharides. Appl. Environ. Microbiol. 74, 4746–4755. Yoshida, A., Yamada, K., Yamazaki, Y., Sashihara, T., Ikegami, S., Shimizu, M., Totsuka, M., 2011. Lactobacillus gasseri OLL2809 and its RNA suppress proliferation of CD4(+) T cells through a MyD88-dependent signalling pathway. Immunology 133, 442–451. Zhang, D., Li, R., Li, J., 2012. Lactobacillus reuteri ATCC 55730 and L22 display probiotic potential in vitro and protect against Salmonella-induced pullorum disease in a chick model of infection. Res. Vet. Sci. 93, 366–373. Zuo, L., Huang, Z., Dong, L., Xu, L., Zhu, Y., Zeng, K., Zhang, C., Chen, J., Zhang, J., 2010. Targeting delivery of anti-TNFalpha oligonucleotide into activated colonic macrophages protects against experimental colitis. Gut 59, 470–479.