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

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.

Figure 1

The cecal microbiota was comprised of bacteria from 10 divisions. A. The Firmicutes and Bacteroidetes phyla comprised approximately 98% of the identified sequences. There was a trend for SDR + 0 hr mice to have higher levels of Firmicutes (p = .08), but lower levels of Bacteroidetes (p = .11) compared to the HCC + 0 hr mice. None of the other groups were found to be significantly different. B. Eight bacterial divisions accounted for the remaining 2% of the identified sequences. There was a trend for SDR + 0 hr mice to have lower levels of Deferribacteres compared to the HCC + 0 hr mice (p = .08). None of the other groups were found to be significantly different. n = 5 per group

Figure 2

Cecal microbiota community structure was different in the SDR + 0 hr mice compared to the HCC + 0 hr mice. The dual hierarchal dendrogram describes the top 40 bacterial genera detected in the SDR + 0 hr and HCC + 0 hr samples. The heat map indicates the relative abundance of the given genus within each sample ID with a color legend and scale provided. The distance of the samples based upon weighted pair linkage and Manhattan distance methods with no scaling is provided at the top of the figure along with a distance score. The bacterial genus and the associated clustering are provided along the y axis, and their associated distance scores are indicated. The dual hierarchical clustering indicates that all 5 SDR + 0 hr samples cluster together, and thus have unique profiles compared to 4 of the 5 HCC + 0 hr samples that clustered together. n = 5 per group.

Figure 3

Cecal microbiota community structure in the SDR + 15 hr and HCC + 15 hr mice. The dual hierarchal dendrogram describes the top 40 bacterial genera detected in the SDR + 15 hr and HCC + 15 hr samples. The heat map indicates the relative abundance of the given genus within each sample ID with a color legend and scale provided. The distance of the samples based upon weighted pair linkage and Manhattan distance methods with no scaling is provided at the top of the figure along with a distance score. The bacterial genus and the associated clustering are provided along the y axis, and their associated distance scores are indicated. The dual hierarchical clustering indicates that 3 of the 5 SDR + 15 hr samples clustered together with, and thus are similar to, 2 of the 5 HCC + 15 hr samples. One SDR + 15 hr sample clustered with the remaining 3 HCC + 15 hr samples, and one SDR + 15 hr sample was found to be unique from the other HCC + 15 hr and SDR + 15 hr samples. n = 5 per group

Figure 4

Exposure to SDR significantly increases circulating levels of IL-6 and MCP-1. A. Mice in the SDR + 0 hr and SDR + 15 hr groups had significantly more IL-6 in their plasma compared to the non-stressed HCC mice at both time points. B. Mice in the SDR + 0 hr group had significantly more MCP-1 than did mice in the HCC + 0 hr group. In all cases * indicates p < .05 vs. HCC at the same time point. n = 5 per group.

Figure 5

Circulating cytokine levels are inversely associated with the relative abundance of cecal microbiota. Circulating IL-6 was inversely associated with the relative abundance of A. Coprococcus spp., B. Pseudobutyrovibrio spp., and C. Dorea spp. D. Circulating MCP-1 was inversely associated with Coprococcus spp. Data are from HCC + 0 hr, HCC + 15 hr, SDR + 0 hr, and SDR + 15 hr animals (n = 5 per group).

Figure 6

Antibiotic treatment prevents the stressor-induced increase in circulating IL-6. A. SDR + 0 hr mice treated with vehicle had significantly higher levels of IL-6 compared to the vehicle treated HCC controls (* p < .05). Giving SDR + 0 hr mice an antibiotic cocktail prevented this stressor-induced increase in IL-6. n = 9 per group. B. SDR + 15 hr mice treated with vehicle had significantly higher levels of IL-6 compared to the vehicle treated HCC controls (* p < .05). Giving SDR + 15 hr mice an antibiotic cocktail prevented this stressor-induced increase in IL-6. n = 9 per group. C. The effects of stressor exposure on circulating IL-6 is not specific to the SDR stressor. The mean level of IL-6 in vehicle-treated mice was increased in mice exposed to a restraint stressor. Antibiotic administration prevented this increase. This increase was not quite statistically significant (p = .14) with this small sample size, n = 5 per group.

Figure 7

Antibiotic administration affects splenic iNOS mRNA. A. The mean level of iNOS mRNA was increased in the spleen immediately after SDR († indicates p = .07). This increased mean level was not evident in the antibiotic treated mice exposed to SDR. B. Antibiotic treatment reduced iNOS mRNA in the spleen regardless of whether mice were in the SDR + 15 hr or HCC + 15 hr group. * indicates main effect for antibiotic administration, p< .05. n = 9 per group.