Rebecca G. Allen

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.

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.

Stressor-Induced Alterations of Adaptive Immunity to Vaccination and Viral Pathogens

The stress response influences the immune system, and studies in laboratory animals indicate that the response to stress significantly reduces resistance to infectious challenge. Only a few studies, however, have determined the impact of the stress response on human susceptibility to infectious challenge due, in part, to the difficulties of using live, replicating pathogens in human research. As a result, many studies have assessed the immune response to vaccination as a surrogate for the immune response to an infectious challenge. Thus, much is known about how the stress response influences adaptive immunity, and memory responses, to vaccination. These studies have yielded data concerning the interactions of the nervous and immune systems and have provided important information for clinicians administering vaccines to susceptible populations. This review provides a brief overview of the immune response to commonly used vaccines and the impact that stress can have on vaccine-specific immunity.

136. Stressor-induced reduction of commensal Lactobacillus reuteri enhances colonic inflammation upon oral challenge with the murine colonic pathogen Citrobacter rodentium

weeks. Following the intervention, mice were i.p. injected with LPS (0.33 mg/kg) or saline. Adipose tissue and brain pro-inflammatory gene expression was analyzed via qRT-PCR. Results: Gene expression analysis revealed a wheel LPS interaction for adipose TNF-alpha (p = .011), indicating wheel training reduced the expression of adipose TNF-alpha post-LPS injection. LPS treatment significantly increased brain TNF-alpha, IL-1beta, and indoleamine 2,3-dioxygenase (IDO) mRNA (p < .05), but there was no wheel LPS interaction at this 24 h time-point. Conclusions: Voluntary-wheel exercise training appears to attenuate LPS-induced adipose inflammation; but this does not translate to a reduction in central inflammation, indicating a disconnect between adipose and brain inflammation following LPS injection. These data suggest adipose tissue inflammation is not a root cause of the exaggerated central inflammatory response observed in aged mice.

130. Repeated social defeat-induced increases in microbicidal activity of splenic macrophages are diminished by inhibiting NO production

from the hippocampus were collected and analyzed by HPLC. Neurotrophin expressions were measured by quantitative PCR. Results: Acute IL-1b injection decreased ACh efflux by 25%, while chronic IL-1 induced a larger decrease (40%), which was partially blocked by IL-1 receptor antagonist or a glucocorticoid receptor antagonist. During learning and memory retrieve, the elevation of ACh efflux was much less in the group treated with chronic IL-1 administration, which was correlated to the memory deficit. Furthermore, acute IL-1b administration increased, while chronic IL-1b decreased BDNF and NGF mRNA expressions in the hippocampus. Conclusion: These results demonstrated that (1) chronic IL-1b impaired memory through reducing ACh releases and neurotrophin expressions, and (2) acute IL-1b may protect the brain by up-regulating neurotrophin expressions.