Metagenome analysis revealed that gut microbiota composition was markedly changed during and after repeated exposure to stressors.
Mice treated with daily 2-h restraint for 14 days, called chronic restraint stress (CRST), exhibit depressive-like behaviors that last longer than 3 months [31, 32]. CRST-induced behavioral deficits are produced by stress-induced maladaptive changes in the hippocampus and neuroendocrine systems [33]. In the present study, we expanded upon previous investigations into the method by which CRST produces changes in gut microbiota.
After they were purchased from a local vendor and habituated in our animal room for 5 days, C57BL6/J mice were randomly divided into two groups: control and stress groups. Mice assigned to stress group were treated with daily 2-h restraint for 14 days and stools were collected at day 1, day 14, and post-stress day 14. Stools were collected from control mice in parallel (Fig. 1A). Bacterial DNA was isolated from fecal samples and used to obtain DNA sequence readings of variable regions of 16S ribosomal RNA (rRNA) genes. This led to the identification of 21,811 and 19,148 operational taxonomic units (OTUs) in the control and CRST groups, respectively. Expected sample taxonomic richness increased with the number of DNA sequence reads in both groups (Fig. 1B). The microbiota identified in CRST and control groups consisted of 22 OTUs at the phylum level, 43 OTUs at the class level, 79 OTUs at the order level, 176 OTUs at the family level, and 376 OTUs at the genus level. Analysis of the relative occupancy of the most abundant 15 phyla with an occupancy ≥ 0.1%, which counted for > 95% of identified OTUs, indicated that the CRST group exhibited significant changes in their % composition relative to the control group over the test period, with a particularly dramatic change at post-stress day 14 (Fig. 1C).
The relative occupancy of the top 15 phyla within a group at each time point was calculated and converted to % composition of the mean value and the mean difference between control group and CRST group, expressed as a standard deviation unit (z score) at each time point. On post-stress day 14, the occupancy of p_Bacteroidetes decreased from 36.90 to 15.33% (-1.95 x z-score), the occupancy of p_Tenericutes decreased from 0.17 to 0.00% (-3.38 x z), and the occupancy of an unassigned phylum decreased from 7.95 to 3.32% (-5.28 x z) (Fig. 1D). In contrast, the occupancy of p_Actinobacteria increased from 4.11 to 12.76% (6.14 x z), the occupancy of p_TM7 increased from 0.07 to 1.50% (56.63 x z), and the occupancy of p_Cyanobacteria increased from 0.11 to 1.09% (18.6 x z) (Fig. 1D).
Chronic stress caused dramatic changes in gut microbiota composition at the genus level.
We next analyzed stress-induced changes in gut microbiota at the genus level. The identified OTUs contained 104 genus members with a relative occupancy of ≥ 0.1% in control or CRST groups at any of the three time points. The most abundant top 10, 20 and 30 genus members in the control group comprised total 88.7, 96.8 and 98.6%, respectively, of those identified OTUs at stress day 1; 91.8, 96.7 and 98.5%, respectively, at stress day 14; and 91.7, 97.2 and 98.7%, respectively, at post-stress day 14 (Fig. 2; Supplemental Table 1). In contrast, those top 10, 20 and 30 genus members in the CRST group comprised 80.8, 92.8 and 96.1%, respectively, of those identified OTUs at stress day 1; 88.0, 92.8 and 96.0%, respectively, at stress day 14; and 22.4, 39.4 and 41.1%, respectively, at post-stress day 14 (Fig. 2; Supplemental Table 1). Thus, the relative abundance of the most abundant top 10–30 genus members in the control group was not changed within a month of the test period, whereas the relative abundance of the same top 10–30 genus members in the CRST group were reduced slightly during the stress period and severely in the post-stress period.
Analysis of the relative occupancy of identified OTUs indicated that 34 genus members had a relative occupancy of ≥ 0.1% in control or CRST groups at any of the three time points. Of those genus members, 6 members were significantly upregulated and 6 members were downregulated in the CRST group over time (time factor, two-way ANOVA), whereas the remaining 22 members were statistically insignificantly changed or unchanged over the test period (Fig. 3; Supplemental Table 1).
Of those altered genus members, the relative occupancy of an unclassified member of f__S24-7 (f__S24-7;g__), an unassigned bacterium, an unclassified member of o_Clostridiales;f_;g_, g__Adlercreutzia and g__Desulfovibrio decreased in the CRST group at post-stress day 14, and the relative occupancy of g__Lactobacillus decreased in the CRST group at all three time points examined. In contrast, the relative occupancy of g__Bacteroides, f__Enterobacteriaceae;g__, f__Comamonadaceae;g__, g__Rhodococcus, g__Pseudomonas, and g__Enhydrobacter increased in the CRST group at post-stress day 14 (Fig. 3; Supplemental Table 1). The relative occupancy of g__Akkermansia, g__Lactococcus, f__Aerococcaceae;g_, g__Ruminococcus and other unclassified member of g__Ruminococcus, g__Faecalibacterium, g__Acinetobacter, o__Streptophyta;f__;g__, g__Parabacteroides, g__Propionibacterium, and g__Blautia appeared to be changed in the CRST group at post-stress day 14, although these differences were not statistically significant (Fig. 3; Supplemental Table 1).
Administration of EVs from three different probiotics to CRST mice induced expression of neurotrophic factors in the hippocampus.
Amongst the taxonomic members of microbiota whose relative abundance was changed by chronic stress, the stress-dependent decrease of Lactobacillus was particularly remarkable (Fig. 3; Supplemental Table 1). Recently, we reported that EVs derived from Lactobacillus plantarum increased Sirt1-dependent Bdnf expression in HT22 cells and in the hippocampus [20]. Consistent with this report, post-stress treatment with EVs from Lactobacillus plantarum rescued the stress-induced decrease in expression of Bdnf and Sirt1 in the hippocampus (Fig. 4A and B). This treatment also reversed stress-induced downregulation of MeCP2 (Fig. 4B), an epigenetic factor which also regulates Bdnf expression [30].
Next, we investigated whether EVs from Bacillus subtilis and Akkermansia muciniphila, which are taxonomically remote from Lactobacillus, could induce similar genomic responses. Post-stress treatment of CRST-treated mice with EVs of Bacillus subtilis and Akkermansia muciniphila also rescued stress-induced expression reduction of Bdnf, Nt3, or Nt4/5. The post-stress treatment with EVs from Bacillus subtilis and Akkermansia muciniphila also increased stress-induced reduced expression of Sirt1, although their changes were not statistically significant (Fig. 4C-F). However, EVs from Bacillus subtilis did not restore stress-induced reduction of MeCP2 expression (Fig. 4D).
EVs from three different types of probiotics all exerted anti-depressant effects.
We next asked whether administration of EVs from those Gram-positive and Gram-negative probiotics could produce behavioral changes in a stress-induced model of depression. EVs from each of the three probiotics described above were administered during the CRST treatment phase via the intraperitoneal route, which permits systemic circulation [34].
Mice exposed to chronic restraint stress (CRST) using daily 2-h restraint for 14 days exhibited increased immobility in the TST and FST (Fig. 5A-C). In contrast, post-stress treatment of CRST-treated mice with EVs of Lactobacillus plantarum for 14 days reversed stress-induced increases in immobility in the TST and FST (Fig. 5A-C).
Post-stress treatment of CRST-treated mice with EVs of Bacillus subtilis and Akkermansia muciniphila for 14 days also rescued stress-induced increases in immobility in the FST, which were comparable to those induced by EVs of Lactobacillus (Fig. 5A-C). However, post-stress treatment of CRST-treated mice with EVs of Bacillus subtilis and Akkermansia muciniphila did not significantly reverse stress-induced increases in immobility in the TST (Fig. 5A-C).
These results suggest that EVs from Lactobacillus plantarum, Bacillus subtilis and Akkermansia muciniphila confer anti-depressant-like effects in CRST-treated mice, although the effects of EVs of Bacillus subtilis and Akkermansia muciniphila are relatively weak.