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 exposure to the SDR stressor enhances the translocation of intestinal, as well as cutaneous, microbiota to secondary lymphoid organs (Bailey et al., 2006
). Although the number of translocating bacteria was low, 47% of the stressor-exposed mice were found to have viable bacteria in the liver (compared to only 14% in the non-stressed controls). Approximately 27% of the translocating bacteria were characterized as Gram-positive bacilli (Bailey et al., 2006
). In the current study, the relative abundance of bacteria in the genus Clostridium
(which are also Gram-positive bacilli) tended to be increased in the SDR + 0 hr mice. Although the increased abundance of clostridia did not result in overt symptomatology (e.g., diarrhea) an overgrowth of microbiota is one factor that can promote bacterial translocation from the gut (Berg, 1999
The SDR stressor significantly reduced microbial diversity as calculated by OTU, ACE, and Chao1 estimators. In addition, using the relative percentage of bacteria classified at the genus level and dual hierarchical clustering methods based on Manhattan distance calculations, it was evident that the overall community structure of the microbiota is different immediately following SDR, but 15 hrs later on the morning following SDR exposure, community structure begins to reflect the profile found in the non-stressed home cage controls. This finding is consistent with other reports indicating that the microbiota are relatively resistant to prolonged alterations (Antonopoulos et al., 2009
). The unique clustering of the SDR + 0 hr samples separately from the other samples is primarily due to an increase in the relative abundance of Clostridium
spp., and lower abundance of Bacteroides
Levels of circulating IL-6 and MCP-1 were directly related to decreases in microbiota abundances, including the abundance of Coprococcus
spp., and Pseudobutyrivibrio
spp. These genera have only recently been described as being part of the human microbiome, and their importance to host physiology is not yet known. However, it is possible that when suppressed, they allow for a bloom in other species, such as the clostridia, that are known to induce inflammation (Brook, 2008
; Libby and Bearman, 2009
). In support of this, there was an inverse relationship between the relative abundance of bacteria in the genus Pseudobutyrovibrio
and the genus Clostridium
(Spearman ρ (8) = − .85, p < .001; data not shown). There was also a trend for Lactobacillus
spp. to be decreased by the stressor, which is consistent with studies showing that stressor exposure suppresses the number of lactobacilli shed from humans and nonhuman primates (Bailey et al., 2004b
; Bailey and Coe, 1999
; Knowles et al., 2008
). Many members of the genus Lactobacillus
can suppress the host inflammatory response (Jones and Versalovic, 2009
; Lin et al., 2008
) and prevent bacterial translocation (Zareie et al., 2006
). Thus, stressor-induced reductions in Psudobutyrivibrio
spp. could allow other members of the microbiota, like the clostridia, to translocate and induce an inflammatory response.
The SDR stressor (Stark et al., 2002
), as well as other laboratory animal stressors like tail shock (Johnson et al., 2005
), is well recognized to induce increases in circulating cytokines and to increase microbicidal activity through the production of reactive nitrogen and oxygen intermediates (Bailey et al., 2007
; Campisi et al., 2002
; Campisi et al., 2003
). Inducible nitric oxide synthase (iNOS) is important for the development of reactive nitrogen and oxygen intermediates, and daily administration of broad spectrum antibiotics significantly reduced splenic iNOS gene expression and circulating IL-6 in the stressor-exposed mice. This effect does not appear to be due to an immunosuppressive effect of the antibiotic, since the dose of antibiotic that was used is below those reported to be immunosuppressive (Fararjeh et al., 2008
), and because injecting antibiotic treated and vehicle treated mice with lipopolysaccharide resulted in similar levels of circulating cytokines (namely, IL-6, MCP-1, IFN-γ, IL-12, and TNF-α) (data not shown). This suggests that baseline levels of microbiota are necessary for stressor-induced increases in inflammatory mediators, and is consistent with other studies that have shown that the microbiota provide a constitutive priming signal to innate immune cells through activation of pattern recognition receptors, particularly the nucleotide-oligomerization domain-containing protein-1 (Nod1) and Nod2 receptors (Clarke et al., 2010
; Kim et al., 2009
). In addition, others have reported that stressor-induced increases in circulating microbial antigen-associated heat shock protein 72 (HSP72) potentiates the innate immune response and enhances iNOS expression (Fleshner et al., 2007
). Our data further emphasize the influence that the microbiota have on the immune system, and indicate that when altered during the stress response, the microbiota influence stressor-induced immunomodulation.
The mechanisms by which the stress response can suppress immune functioning are well understood, and are known to involve glucocorticoid mediated suppression of transcription factors, such as NF-κB (Padgett and Glaser, 2003
). However, the mechanisms by which the stress response enhances immune activity are not yet fully elucidated. Recent studies indicate that the sympathetic nervous system (SNS) is involved in stressor-induced immune enhancement (Bierhaus et al., 2003
; Cole et al., 2010
), and blocking the activation of the SNS through the use of the α- and β-adrenergic receptor antagonists has been reported to block the stressor-induced increases in innate immunity (Hanke et al., 2008
; Johnson et al., 2005
). In addition to direct effects on the immune system, the SNS is also involved in the bidirectional communication between the gut and the brain that facilitates the regulation of gut function. Stressor-induced SNS activity significantly impacts gut secretion and motility (Lomax et al., 2010
), which can in turn impact the stability of the microbiota. In addition, SNS-derived catecholamines, particularly norepinephrine (NE) have the capacity to stimulate the growth of many enteric bacteria (Freestone et al., 2000
; Lyte et al., 1997
; Lyte, 2004
), including Clostridium
spp. (Lyte, 2004
) which were found to be increased in the SDR-exposed mice. Thus, it is possible that stressor-induced SNS activity directly and/or indirectly affects the microbiota and results in increased reactivity of the innate immune system.
It is well recognized that stressor exposure is associated with elevated circulating inflammatory cytokines. Studies in humans demonstrate that both prolonged natural stressors, as well as acute laboratory stressors result in increased circulating cytokines such as IL-6 and TNF-α (reviewed in (Steptoe et al., 2007
). Whether stressor-induced cytokines in humans are related to stressor-induced effects on the microbiota is not known, but studies have indicated that humans experiencing stressful situations have altered profiles of intestinal microbiota (Holdeman et al., 1976
; Knowles et al., 2008
). And, bacterial translocation, as assessed by an increased occurrence of circulating antibodies to microbiota, has been linked to mood disorders, such as depression, presumaby through a cytokine-mediated mechanism (Maes, 2008
; Maes et al., 2008
). This is consistent with findings in patients with functional and inflammatory bowel disease that have frequent psychiatric co-morbidity and alterations in the intestinal microbiota (Collins and Bercik, 2009
). Our studies indicate the microbiota are primarily and interactively involved in stressor-induced immunoenhancement and contribute to the growing literature on the impact that the microbiota have on the health of the host.
- 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 3 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.