Michael T. Bailey

Exposure to a Social Stressor Alters the Structure of the Intestinal Microbiota: Implications for Stressor-Induced Immunomodulation

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.

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

The integrity of the indigenous microflora of the intestines after maternal separation was investigated in infant rhesus monkeys to determine whether psychological stress may lead to an internal environment conducive to pathogen infection. The stability of the indigenous microflora were estimated by enumeration of total and gram-negative aerobic and facultatively anaerobic bacterial species, specifically Lactobacilli, from coprocultures taken before and after maternal separation. In addition, behavioral and cortisol responses to separation were correlated to the microflora. A significant decrease in fecal bacteria, especially Lactobacilli, was evident on day 3 postseparation, with a return to baseline by the end of the week. The drop in the microflora was correlated with the display of stress-indicative behaviors, but not with cortisol secretion. In addition, infants who displayed numerous stress-indicative behaviors were more susceptible to opportunistic bacterial infection. These results suggest that strong emotional reactions to disruption of the mother-infant bond may increase vulnerability to disease.

Stressor Exposure Disrupts Commensal Microbial Populations in the Intestines and Leads to Increased Colonization by Citrobacter rodentium

ABSTRACT The gastrointestinal tract is colonized by an enormous array of microbes that are known to have many beneficial effects on the host. Previous studies have indicated that stressor exposure can disrupt the stability of the intestinal microbiota, but the extent of these changes, as well as the effects on enteric infection, has not been well characterized. In order to examine the ability of stressors to induce changes in the gut microbiota, we exposed mice to a prolonged restraint stressor and then characterized microbial populations in the intestines using both traditional culture techniques and bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Exposure to the stressor led to an overgrowth of facultatively anaerobic microbiota while at the same time significantly reducing microbial richness and diversity in the ceca of stressed mice. Some of these effects could be explained by a stressor-induced reduction in the relative abundance of bacteria in the family Porphyromonadaceae. To determine whether these alterations would lead to increased pathogen colonization, stressed mice, as well as nonstressed controls, were challenged orally with the enteric murine pathogen Citrobacter rodentium. Exposure to the restraint stressor led to a significant increase in C. rodentium colonization over that in nonstressed control mice. The increased colonization was associated with increased tumor necrosis factor alpha (TNF-α) gene expression in colonic tissue. Together, these data demonstrate that a prolonged stressor can significantly change the composition of the intestinal microbiota and suggest that this disruption of the microbiota increases susceptibility to an enteric pathogen.

Prenatal stress alters bacterial colonization of the gut in infant monkeys.

Objective: The hypothesis that prenatal stress lowers the levels of protective microflora and increases the risk for postpartum Gram-negative pathogens was tested in infant monkeys. Methods: Female monkeys were left undisturbed or were stressed during pregnancy using an acoustical startle paradigm for 6 weeks either early or late in their 24-week gestation. Several types of intestinal microflora were repeatedly enumerated by fecal culture while infants were reared normally by their mothers. Results: Significant changes in microflora concentrations occurred during the first 6 months of life. The profile of total aerobes and facultative anaerobes was biphasic, with peak concentrations occurring between 2 and 16 weeks of age. The numbers of bifidobacteria and lactobacilli were low at 2 days after birth but rapidly increased to a peak between 8 and 16 weeks of age. Although similar temporal patterns were evident in all infants, prenatal stress reduced the overall numbers of bifidobacteria and lactobacilli. Conclusions: Moderate disturbance during pregnancy was sufficient to alter the intestinal microflora in the newborn infant. These alterations could result in enhanced susceptibility to infection and suggest a mechanism for some effects of maternal pregnancy conditions on infant health.

Beta adrenergic blockade decreases the immunomodulatory effects of social disruption stress

During physiological or psychological stress, catecholamines produced by the sympathetic nervous system (SNS) regulate the immune system. Previous studies report that the activation of β-adrenergic receptors (βARs) mediates the actions of catecholamines and increases pro-inflammatory cytokine production in a number of different cell types. The impact of the SNS on the immune modulation of social defeat has not been examined. The following studies were designed to determine whether SNS activation during social disruption stress (SDR) influences anxiety-like behavior as well as the activation, priming, and glucocorticoid resistance of splenocytes after social stress. CD-1 mice were exposed to one, three, or six cycles of SDR and HPLC analysis of the plasma and spleen revealed an increase in catecholamines. After six cycles of SDR the open field test was used to measure behaviors characteristic of anxiety and indicated that the social defeat induced increase in anxiety-like behavior was blocked by pre-treatment with the β-adrenergic antagonist propranolol. Pre-treatment with the β-adrenergic antagonist propranolol did not significantly alter corticosterone levels indicating no difference in activation of the hypothalamic-pituitary-adrenal axis. In addition to anxiety-like behavior the SDR induced splenomegaly and increase in plasma IL-6, TNFα, and MCP-1 were each reversed by pre-treatment with propranolol. Furthermore, flow cytometric analysis of cells from propranolol pretreated mice reduced the SDR-induced increase in the percentage of CD11b(+) splenic macrophages and significantly decreased the expression of TLR2, TLR4, and CD86 on the surface of these cells. In addition, supernatants from 18h LPS-stimulated ex vivo cultures of splenocytes from propranolol-treated SDR mice contained less IL-6. Likewise propranolol pre-treatment abrogated the glucocorticoid insensitivity of CD11b(+) cells ex vivo when compared to splenocytes from SDR vehicle-treated mice. Together, this study demonstrates that the immune activation and priming effects of SDR result, in part, as a consequence of SNS activation.

Probiotic Lactobacillus reuteri Attenuates the Stressor-Enhanced Severity of Citrobacter rodentium Infection

ABSTRACT Stressor exposure has been shown to enhance host susceptibility and the severity of a plethora of illnesses, including gastrointestinal disease. In mice, susceptibility to Citrobacter rodentium has been shown to be dependent on host genetics as well as the composition of the intestinal microbiota, but the effects of stressor exposure on this gastrointestinal pathogen have not been elucidated fully. Previously, our lab showed that exposure to the prolonged-restraint stressor prior to a challenge with C. rodentium alters the intestinal microbiota community structure, including a reduction of beneficial genera such as Lactobacillus, which may contribute to stressor-enhanced C. rodentium-induced infectious colitis. To test the effects of stressor exposure on C. rodentium infection, we exposed resistant mice to a prolonged-restraint stressor concurrent with pathogen challenge. Exposure to prolonged restraint significantly enhanced C. rodentium-induced infectious colitis in resistant mice, as measured by increases in colonic histopathology, colonic inflammatory mediator gene production, and pathogen translocation from the colon to the spleen. It was further tested if the beneficial bacterium Lactobacillus reuteri could reduce the stressor-enhanced susceptibility to C. rodentium-enhanced infectious colitis. While L. reuteri treatment did not reduce all aspects of stressor-enhanced infectious colitis, it did significantly reduce pathogen translocation from the colon to the spleen. Taken together, these data demonstrate the deleterious effects that prolonged stressor exposure can have at the onset of a gastrointestinal infection by its ability to render a resistant mouse highly susceptible to C. rodentium. Probiotic treatment ameliorated the systemic manifestations of stress on colonic infection.

The Intestinal Microbiota Are Necessary for Stressor-Induced Enhancement of Splenic Macrophage Microbicidal Activity

The indigenous microbiota impact mucosal, as well as systemic, immune responses, but whether the microbiota are involved in stressor-induced immunomodulation has not been thoroughly tested. A well characterized murine stressor, called social disruption (SDR), was used to study whether the microbiota are involved in stressor-induced enhancement of macrophage reactivity. Exposure to the SDR Stressor enhanced the ability of splenic macrophages to produce microbicidal mediators (e.g., inducible nitric oxide synthase (iNOS), superoxide anion, and peroxynitrite) and to kill target Escherichia coli. Exposure to the SDR Stressor also increased cytokine production by LPS-stimulated splenic macrophages. These effects, however, were impacted by the microbiota. Microbicidal activity and cytokine mRNA in splenic macrophages from Swiss Webster germfree mice that lack any commensal microbiota were not enhanced by exposure to the SDR Stressor. However, when germfree mice were conventionalized by colonizing them with microbiota from CD1 conventional donor mice, exposure to the SDR Stressor again increased microbicidal activity and cytokine mRNA. In follow-up experiments, immunocompetent conventional CD1 mice were treated with a cocktail of antibiotics to disrupt the intestinal microbiota. While exposure to the SDR Stressor-enhanced splenic macrophage microbicidal activity and cytokine production in vehicle-treated mice, treatment with antibiotics attenuated the SDR Stressor-induced increases in splenic macrophage reactivity. Treatment with antibiotics also prevented the stressor-induced increase in circulating levels of bacterial peptidoglycan, suggesting that translocation of microbiota-derived peptidoglycan into the body primes the innate immune system for enhanced activity. This study demonstrates that the microbiota play a crucial role in stressor-induced immunoenhancement.

Stressor-Induced Increase in Microbicidal Activity of Splenic Macrophages Is Dependent upon Peroxynitrite Production

ABSTRACT Exposing mice to a social stressor called social disruption (SDR) that involves repeated social defeat during intermale aggression results in increased circulating cytokines, such as interleukin-1α (IL-1α) and IL-1β, and increased reactivity of splenic CD11b+ macrophages to inflammatory stimuli. For example, upon lipopolysaccharide stimulation, macrophages from stressor-exposed mice produce higher levels of cytokines than do cells from nonstressed controls. Moreover, the SDR stressor enhances the ability of these macrophages to kill Escherichia coli both in vitro and in vivo, through a Toll-like receptor 4-dependent mechanism. The present study tested the hypothesis that stressor-enhanced bacterial killing is due to increases in the production of peroxynitrite. Male mice were exposed to the SDR stressor or were left undisturbed. Upon stimulation with E. coli, splenic macrophages from SDR-exposed mice expressed significantly increased levels of inducible nitric oxide synthase mRNA and produced higher levels of peroxynitrite. Blocking the production of peroxynitrite abrogated the SDR-induced increase in microbicidal activity. Studies in IL-1 receptor type 1 knockout mice indicated that the increased microbicidal activity and peroxynitrite production was dependent upon IL-1 signaling. These data confirm and extend the importance of IL-1 signaling for stressor-induced immunopotentiation; the finding that inhibiting superoxide or nitric oxide production inhibits both peroxynitrite production and killing of E. coli demonstrates that peroxynitrite mediates the stressor-induced increase in bacterial killing.

In Vivo Adaptation of Attenuated Salmonella typhimurium Results in Increased Growth Upon Exposure to Norepinephrine

Two studies were conducted to determine whether attenuated strains of Salmonella typhimurium, currently being investigated as possible vectors for mucosal vaccines, are able to respond to norepinephrine (NE). Bacteria were tested for NE responsiveness before and for 1 week after passage through juvenile rhesus monkeys. NE significantly increased the growth of the attenuated bacteria after being shed from the animal, but not before animal infection. Follow-up in vitro tests were performed by passaging the bacteria in Lauria-Bertani (LB) broth with or without selective antibiotic for the attenuation insert and supplementing with NE. NE increased the growth of bacteria passaged in LB broth with no selective antibiotic, but not in bacteria passaged in LB broth with selective antibiotic. These results show that the attenuated bacteria assumed to be safe for use as a vaccine are able to respond to environmental stimuli, such as NE, and change their characteristics. The results suggest that there may be problems with the stability of attenuated bacteria used as vectors for mucosal vaccines.

Intestinal microbial patterns of the common marmoset and rhesus macaque.

The intestinal microflora of common marmosets and rhesus monkeys were compared by enumerating bacteria from the small and large intestines. Rhesus monkeys had a consistent microflora pattern manifest by higher concentrations of total and Gram-negative aerobic and facultatively anaerobic bacteria, as well as aerobic and anaerobic Lactobacilli, in the large intestine as compared to the small intestine. In contrast, the marmoset microflora were considerably more variable. Approximately two-thirds of the marmosets (designated group A) had an overall profile that resembled the rhesus monkeys, but they had significantly higher concentrations of Gram-negative microflora in their large intestines than the rhesus monkeys. The remaining marmosets (group B) had higher concentrations of bacteria in the small intestine as compared to the large intestine, with the large intestinal concentrations being significantly lower than in the rhesus monkeys and group A marmosets. Moreover, the marmosets did not have detectable levels of aerobic Lactobacilli, and anaerobic Lactobacilli concentrations were significantly lower than in the rhesus macaques. Although it is unknown why microflora differ across species, it is likely that evolutionary adaptations in anatomy and functioning of the gastrointestinal tract influence the concentration and types of bacteria residing as the normal intestinal microflora.