Antidepressants affect gut microbiota and Ruminococcus flavefaciens is able to abolish their effects on depressive-like behavior

Abstract

Accumulating evidence demonstrates that the gut microbiota affects brain function and behavior, including depressive behavior. Antidepressants are the main drugs used for treatment of depression. We hypothesized that antidepressant treatment could modify gut microbiota which can partially mediate their antidepressant effects. Mice were chronically treated with one of five antidepressants (fluoxetine, escitalopram, venlafaxine, duloxetine or desipramine), and gut microbiota was analyzed, using 16s rRNA gene sequencing. After characterization of differences in the microbiota, chosen bacterial species were supplemented to vehicle and antidepressant-treated mice, and depressive-like behavior was assessed to determine bacterial effects. RNA-seq analysis was performed to determine effects of bacterial treatment in the brain. Antidepressants reduced richness and increased beta diversity of gut bacteria, compared to controls. At the genus level, antidepressants reduced abundances of Ruminococcus, Adlercreutzia, and an unclassified Alphaproteobacteria. To examine implications of the dysregulated bacteria, we chose one of antidepressants (duloxetine) and investigated if its antidepressive effects can be attenuated by simultaneous treatment with Ruminococcus flavefaciens or Adlercreutzia equolifaciens. Supplementation with R. flavefaciens diminished duloxetine-induced decrease in depressive-like behavior, while A. equolifaciens had no such effect. R. flavefaciens treatment induced changes in cortical gene expression, up-regulating genes involved in mitochondrial oxidative phosphorylation, while down-regulating genes involved in neuronal plasticity. Our results demonstrate that various types of antidepressants alter gut microbiota composition, and further implicate a role for R. flavefaciens in alleviating depressive-like behavior. Moreover, R. flavefaciens affects gene networks in the brain, suggesting a mechanism for microbial regulation of antidepressant treatment efficiency.

Fig. 1. Antidepressants alter diversity of gut microbiota.

a Experimental design of the study examining antidepressant effects on gut microbiota. b, c Measures of alpha diversity. All antidepressants, except desipramine, reduced richness of microbial communities (PD whole tree is shown) (b), but there was no changes in the evenness (Gini coeficient is shown) (c). d–f Evaluations of beta diversity. Bacterial communities of mice treated with antidepressants displayed higher unweighted UniFrac distances in comparison with controls (d) and microbial communities of control group were more similar to each other than to the communities of antidepressant treated mice (e). Unweighted UniFrac-based principal coordinates analysis (PCoA) plot used to visualize microbial communities of all antidepressant treated and control mice (the percentage of variation explained by the principal coordinates is indicated on the axes) (f). * p < 0.05, ** p < 0.01, FDR corrected, nonparametric t-tests with 999 Monte Carlo permutations in comparison to control group; n = 9 (control), n = 11 (flu), n = 12 (esc), n = 12 (ven), n = 11 (dul), n = 12 (des), animals per group; data represent mean ± SEM. flu fluoxetine, esc escitalopram, ven venlafaxine, dull duloxetine, des desipramine

Fig. 2. Bacterial taxa changed by antidepressants.

a, b Results of LEfSe analyses. Taxonomic cladograms representing bacterial taxa differently abundant in stool samples from different groups: VIOLET- bacterial taxa more abundant in control group, compared to all antidepressant treated groups; RED - bacterial taxa more abundant in fluoxetine treated mice; GREEN - bacterial taxa more abundant in escitalopram treated mice; BLUE - bacterial taxa more abundant in desipramine treated mice. There were no bacterial taxa that were more abundant in venlafaxine or duloxetine treated mice compared to all other groups (a). Visualization of bacterial taxa, ranked by effect size, that were more abundant in control group compared to all antidepressant treated groups (p < 0.05, LDA>2) (b). c–f Validation of Ruminococcus and Adlercreutzia levels. Reduced relative abundance of OTU 228330, assigned to species Ruminococcus flavefaciens (c) and reduced levels of Ruminococcus flavefaciens quantified by qRT-PCR (d) in stool samples of antidepressant treated mice compared to controls. Reduced relative abundance of OTU 245324, with 98% of similarity to Adlercreutzia equolifaciens (e), and reduced levels of Adlercreutzia equolifaciens quantified by qRT-PCR, in stool samples of antidepressant treated mice compared to controls (f). Levels of gut bacteria were normalized to control group. ^# 0.1 > p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney tests in comparison to control group, followed by FDR correction; n = 9 (control), n = 11 (flu), n = 12 (esc), n = 12 (ven), n = 11 (dul), n = 12 (des), animals per group; data represent mean ± SEM. flu fluoxetine, esc escitalopram, ven venlafaxine, dul duloxetine, des desipramine

Fig. 3. R. flavefaciens, but not A. equolifaciens, abolished antidepressive effect of duloxetine.

a Experimental design of studies examining behavioral effects of duloxetine and bacterial treatments (R. flavefaciens or A. equolifaciens). Mice were chronically treated with antidepressant and/or bacteria, followed by behavioral testing. b–d Effects of duloxetine and R. flavefaciens on depressive-like behavior. R. flavefaciens supplementation abolished antidepressive effect of duloxetine in tail suspension test (b), forced swim test (c) and sucrose preference test (d). n = 10 (control), n = 10 (dul), n = 11 (Rum), n = 11 (dul+Rum), animals per group. e, f Effects of duloxetine and A. equolifaciens on depressive-like behavior. Duloxetine still reduced depressive-like behavior after A. equolifaciens treatment in tail suspension test (e) and forced swim test (f). n = 10 (control), n = 10 (dul), n = 11 (Adl), n = 10 (dul+Adl), animals per group. ^#0.1 > p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, Tukey’s post hoc test; data represent mean ± SEM. dul duloxetine, Rum R. flavefaciens, Adl A. equolifaciens

Fig. 4. Differentially expressed genes (DEGs) after duloxetine and R. flavefaciens treatments.

a The total number of DEGs, as well as the number of genes that are up-regulated and down-regulated by the particular treatment, are represented in the table. b, c Gene ontology (GO) enrichment analyses of genes up-regulated (b) and down-regulated (c) by R. flavefaciens treatment. d, e GO enrichment analyses of genes up-regulated (d) and down-regulated (E) by concomitant duloxetine and R. flavefaciens treatment. Bars representing GO terms show Benjamini and Hochberg FDR adjusted p values. n = 6 animals per experimental group. dul duloxetine, Rum R. flavefaciens

Fig. 5. Results of weighted gene correlation network analysis (WGCNA).

a Table of module-trait relationship reports Kendall’s correlation coefficients, and its corresponding p values, between the eigengene value of each module and the particular treatments. Modules related to R. flavefaciens treatment, and were not correlated with the same directionality with duloxetine treatment, are emphasized by dashed lines. b–g Gene ontology (GO) and protein-protein interaction (PPI) network analysis of WGCNA modules significantly related to R. flavefaciens treatment. GO enrichment analyses (b) and PPI network analysis (c, d) of genes in blue module with module membership (MM) > 0.7. GO enrichment analyses (e) and PPI network analysis (f) of genes in turquoise module with MM > 0.7. GO enrichment analyses of genes in skyblue module, with MM > 0.7 (g) Bars representing GO terms show Benjamini and Hochberg FDR adjusted p values. Node size in PPI networks indicates number of interactions with other nodes in the network (i.e., degree centrality), while the node color reflects number of shortest paths that rely on that given node within the network (i.e., betweenness centrality). dul duloxetine, Rum R. flavefaciens