Exposure to a social stressor alters the structure of the intestinal microbiota: Implications for stressor-induced immunomodulation

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Abstract

The bodies of most animals are populated by highly complex and genetically diverse communities of microorganisms. The majority of these microbes reside within the intestines in largely stable but dynamically interactive climax communities that positively interact with their host. Studies from this laboratory have shown that stressor exposure impacts the stability of the microbiota and leads to bacterial translocation. The biological importance of these alterations, however, is not well understood. To determine whether the microbiome contributes to stressor-induced immunoenhancement, mice were exposed to a social stressor called social disruption (SDR), that increases circulating cytokines and primes the innate immune system for enhanced reactivity. Bacterial populations in the cecum were characterized using bacterial tag-encoded FLX amplicon pyrosequencing. Stressor exposure significantly changed the community structure of the microbiota, particularly when the microbiota were assessed immediately after stressor exposure. Most notably, stressor exposure decreased the relative abundance of bacteria in the genus Bacteroides, while increasing the relative abundance of bacteria in the genus Clostridium. The stressor also increased circulating levels of IL-6 and MCP-1, which were significantly correlated with stressor-induced changes to three bacterial genera (i.e., Coprococcus, Pseudobutyrivibrio, and Dorea). In follow up experiments, mice were treated with an antibiotic cocktail to determine whether reducing the microbiota would abrogate the stressor-induced increases in circulating cytokines. Exposure to SDR failed to increase IL-6 and MCP-1 in the antibiotic treated mice. These data show that exposure to SDR significantly affects bacterial populations in the intestines, and remarkably also suggest that the microbiota are necessary for stressor-induced increases in circulating cytokines.

Research highlights

► Exposure to the social stressor, called social disruption, significantly changed the community structure of the intestinal microbiota. ► Stressor-induced increases in circulating IL-6 and MCP-1 were significantly correlated with stressor-induced changes in three members of the microbiota, Dorea spp., Coprococcus spp., and Pseudobutyrivibrio spp. ► Administration of a broad spectrum antibiotic cocktail to reduce the microbiota prevented the stressor-induced increase in IL-6 and splenic inducible nitric oxide synthase gene expression.

Introduction

The external surfaces of the body are colonized by vast arrays of microbes that outnumber cells of the body by a factor of 10 (i.e., 1014 bacterial cells to 1013 human cells). This means that 90% of the cells of our body are our commensal microbiome. The majority of these microbes reside in the intestines as part of the intestinal microbiota, with microbiota levels ranging from <105 bacteria per gram of digesta in the upper parts of the small intestine, to >1012 bacteria per gram of digesta in the large intestine (Sekirov et al., 2010). Many of these bacteria are simple opportunistic colonizers, while the majority are true symbiotic organisms in the sense that they have beneficial interactions with each other and the host. For example, many metabolic activities in the intestines are derived from the microbiota, such as the synthesis of vitamin K and vitamin B complex and the metabolism of carcinogens (O’Hara and Shanahan, 2006). In addition, studies utilizing germ-free mice have now linked changes in the intestinal microbiota to the development of obesity and diabetes (Backhed et al., 2004, Ley et al., 2005, Turnbaugh et al., 2006). Perhaps one of the most well studied effects of the microbiota on the host, however, is their impact on the immune system.

It has long been recognized that the intestinal microbiota affect the mucosal immune system. This has been well documented in germ-free animals that due to a lack of commensal microbiota have smaller Peyer’s patches, fewer intraepithelial lymphocytes, and lower levels of secretory IgA (Macpherson and Uhr, 2004). Importantly, colonizing the germ-free mice with commensal bacteria normalizes these immune parameters, indicating the importance of intestinal bacteria in shaping the mucosal immune response (Macpherson and Uhr, 2004). The impact that the microbiota can have on systemic immunity has not been as widely studied. But, alterations to the intestinal microbiota have been linked to inflammatory diseases, such as asthma, in animal models and also in humans, suggesting that the microbiota affect aspects of adaptive immune regulation (Huffnagle, 2010). Innate immunity has also been shown to be affected by the microbiota, with studies indicating that neutrophil activity is primed in vivo by microbiota-derived peptidoglycan acting on neutrophil nucleotide oligomerization domain-containing protein (Nod)1 receptors (Clarke et al., 2010). Moreover, translocation of intestinal bacteria from the lumen of the intestines to the interior of the body has been shown to result in increases in circulating cytokines like IL-6 (Ando et al., 2000). Thus, there is ample evidence linking the microbiota to innate and adaptive immune responses at mucosal, as well as systemic, sites.

The microbiota reside as a largely stable climax community as a result of a series of ecological successions involving the selection of species best adapted for the given niche (Huffnagle, 2010). This climax community is resistant and resilient to long-term disruptions in community structure (Allison and Martiny, 2008), but many factors, such as diet or antibiotic use, can cause more transient alterations in their community structure (Antonopoulos et al., 2009, Dethlefsen et al., 2008). Studies from our laboratories, and from others, demonstrate that stressor exposure, or exposure to neuroendocrine hormones, can significantly affect the microbiota (Bailey et al., 2010, Bailey and Coe, 1999, Knowles et al., 2008, Lizko, 1987, Lyte and Bailey, 1997, Tannock and Savage, 1974). A study using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) demonstrated that the community structure of microbiota from mice exposed to a prolonged restraint stressor was significantly different than the community structure found in non-stressed control mice (Bailey et al., 2010). The importance of these findings for the health of the host is not completely understood. While there is evidence to suggest that changes in the intestinal microbiota reduces resistance to infectious challenge with intestinal pathogens (Bailey et al., 2010), it is also known that altering the climax communities of microbiota is a predisposing factor for translocation of bacteria from the lumen of the intestines to the interior of the body (Berg, 1999) where they can stimulate the immune system (Clarke et al., 2010, Kim et al., 2009). Although we have reported that stressor exposure increases the translocation of gastrointestinal and cutaneous microbiota to secondary lymphoid organs (Bailey et al., 2006), the likelihood that stressor-induced effects on the microbiota also impact stressor-induced immunomodulation has not been tested.

Psychological stressors in humans, and in laboratory animals, often cause an increase in circulating cytokines. For example, prolonged stressors, such as the stress of caring for a spouse with dementia or chronic work stress, are associated with elevated cytokines like IL-6 and TNF-α (Hemingway et al., 2003, Kiecolt-Glaser et al., 2003). Acute laboratory stressors, like the Trier social stress test (TSST) and the Stroop task also cause elevated circulating IL-6 and TNF-α (Brydon et al., 2004, Brydon et al., 2005, Brydon and Steptoe, 2005, Edwards et al., 2006, von et al., 2006). In laboratory mice, repeated social defeat incurred during stressor paradigms including social conflict and social disruption (SDR) results in enhanced innate immune activity (Bailey et al., 2007, Bailey et al., 2009, Lyte et al., 1990, Powell et al., 2009). Cytokines, including IL-6, are increased in the circulation of mice exposed to the SDR paradigm (Stark et al., 2002), and splenic macrophages from the repeatedly defeated mice are primed for enhanced cytokine production and antimicrobial activity upon bacterial stimulation (Bailey et al., 2007, Bailey et al., 2009). The mechanisms linking the stress response to increases in immune function are not well understood, but evidence suggests that stressor-induced increases in sympathetic nervous system (SNS) activity can enhance innate immune activity (Bierhaus et al., 2003). Because stressor exposure changes the community structure of the microbiota (Bailey et al., 2010), and induces bacterial translocation (Bailey et al., 2006), and because microbiota products have been shown to be necessary for priming of innate immunity (Clarke et al., 2010), we hypothesized that microbiota are an important component of the stressor-induced enhancement of innate immune activity. To test this hypothesis, we first determined whether exposure to the SDR stressor changed the community structure of the microbiota. This was followed by experiments in which antibiotics were given to stressor-exposed, as well as non-stressed control, mice to determine whether reducing the microbiota would abrogate stressor-induced increases in immune activity.

Section snippets

Animals

Male CD-1 mice, 6–8 weeks of age, were purchased from Charles River Laboratories (Wilmington, MA) and allowed to acclimate to the animal vivarium for 1 week prior to experimentation. The mice were housed in groups of 3–5 per cage and were kept on a 12 h light:dark schedule with lights on at 0600. Food and water were available ad libitum. All experimental procedures were approved by The Ohio State University’s Animal Care and Use Committee.

Social disruption

The social disruption (SDR) stressor occurred over a 2 h

Impact of stress exposure on the intestinal microbiota

Exposure to the SDR stressor significantly affected the intestinal microbiota. This was manifest as a reduction in microbial diversity and richness. Mathematical evaluation of diversity and richness estimates (at 3% and 5% divergence, which roughly correspond to species and genus levels) based upon Ace and Chao1, as well as curve fitting Richard’s equations to predict maximum potential operational taxonomic units indicated a significant reduction in microbial diversity in the SDR + 15 h group

Discussion

The results of this study indicate that reducing the indigenous microbiota blocks the stressor-induced increase in circulating IL-6 and iNOS mRNA in the spleen. Although the mechanisms through which this occurs have not yet been systematically studied, it is likely that stressor-induced alterations of the microbiota results in translocation of bacteria and/or bacterial products across the intestinal barrier to act as a priming stimulus for the innate immune system. We previously reported that

Acknowledgments

The authors would like to thank Dr. Mark Hanke for assistance with CBA data analysis. This work was supported by NIH RO3AI069097-01A1 and Ohio State University start up funds to M.T.B., and by Texas Tech University Health Sciences Center internal grants to M.L. R.G.A. and A.R.H. were supported via T32 training grant DE014320.

References (67)

  • M. Fararjeh et al.

    Evaluation of immunosuppression induced by metronidazole in Balb/c mice and human peripheral blood lymphocytes

    Int. Immunopharmacol.

    (2008)
  • M.L. Hanke et al.

    Beta-2 adrenergic blockade decreases the immunomodulatory effects of Social Disruption Stress (SDR)

    Brain Behav. Immun.

    (2008)
  • H. Hemingway et al.

    Social and psychosocial influences on inflammatory markers and vascular function in civil servants (the Whitehall II study)

    Am. J. Cardiol.

    (2003)
  • J.D. Johnson et al.

    Catecholamines mediate stress-induced increases in peripheral and central inflammatory cytokines

    Neuroscience

    (2005)
  • S.R. Knowles et al.

    Investigating the role of perceived stress on bacterial flora activity and salivary cortisol secretion: a possible mechanism underlying susceptibility to illness

    Biol. Psychol.

    (2008)
  • D.B. Libby et al.

    Bacteremia due to Clostridium difficile – review of the literature

    Int. J. Infect. Dis.

    (2009)
  • M. Lyte

    Microbial endocrinology and infectious disease in the 21st century

    Trends Microbiol.

    (2004)
  • M. Lyte et al.

    Neuroendocrine–bacterial interactions in a neurotoxin-induced model of trauma

    J. Surg. Res.

    (1997)
  • M. Lyte et al.

    Innate and adaptive immune responses in a social conflict paradigm

    Clin. Immunol. Immunopathol.

    (1990)
  • D.A. Padgett et al.

    How stress influences the immune response

    Trends Immunol.

    (2003)
  • N.D. Powell et al.

    Repeated social defeat activates dendritic cells and enhances Toll-like receptor dependent cytokine secretion

    Brain Behav. Immun.

    (2009)
  • S. Rakoff-Nahoum et al.

    Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis

    Cell

    (2004)
  • J.L. Stark et al.

    Interleukin-6 and the development of social disruption-induced glucocorticoid resistance

    J. Neuroimmunol.

    (2002)
  • A. Steptoe et al.

    The effects of acute psychological stress on circulating inflammatory factors in humans: a review and meta-analysis

    Brain Behav. Immun.

    (2007)
  • S.D. Allison et al.

    Colloquium paper: resistance, resilience, and redundancy in microbial communities

    Proc. Natl. Acad. Sci. USA

    (2008)
  • T. Ando et al.

    Bacterial translocation can increase plasma corticosterone and brain catecholamine and indoleamine metabolism

    Am. J. Physiol. Regul. Integr. Compar. Physiol.

    (2000)
  • D.A. Antonopoulos et al.

    Reproducible community dynamics of the gastrointestinal microbiota following antibiotic perturbation

    Infect. Immun.

    (2009)
  • F. Backhed et al.

    The gut microbiota as an environmental factor that regulates fat storage

    Proc. Natl. Acad. Sci. USA

    (2004)
  • M.T. Bailey et al.

    Maternal separation disrupts the integrity of the intestinal microflora in infant rhesus monkeys

    Dev. Psychobiol.

    (1999)
  • M.T. Bailey et al.

    Stressor exposure disrupts commensal microbial populations in the intestines and leads to increased colonization by Citrobacter rodentium

    Infect. Immun.

    (2010)
  • M.T. Bailey et al.

    Repeated social defeat increases the bactericidal activity of splenic macrophages through a Toll-like receptor-dependent pathway

    Am. J. Physiol. Regul. Integr. Compar. Physiol.

    (2007)
  • M.T. Bailey et al.

    Prenatal stress alters bacterial colonization of the gut in infant monkeys

    J. Pediatr. Gastroenterol. Nutr.

    (2004)
  • R.D. Berg

    Bacterial translocation from the gastrointestinal tract

    Adv. Exp. Med. Biol.

    (1999)
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