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. 2015 Apr 9;161(2):264-76.
doi: 10.1016/j.cell.2015.02.047.

Indigenous Bacteria From the Gut Microbiota Regulate Host Serotonin Biosynthesis

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Free PMC article

Indigenous Bacteria From the Gut Microbiota Regulate Host Serotonin Biosynthesis

Jessica M Yano et al. Cell. .
Free PMC article

Erratum in

  • Cell. 2015 Sep 24;163:258

Abstract

The gastrointestinal (GI) tract contains much of the body's serotonin (5-hydroxytryptamine, 5-HT), but mechanisms controlling the metabolism of gut-derived 5-HT remain unclear. Here, we demonstrate that the microbiota plays a critical role in regulating host 5-HT. Indigenous spore-forming bacteria (Sp) from the mouse and human microbiota promote 5-HT biosynthesis from colonic enterochromaffin cells (ECs), which supply 5-HT to the mucosa, lumen, and circulating platelets. Importantly, microbiota-dependent effects on gut 5-HT significantly impact host physiology, modulating GI motility and platelet function. We identify select fecal metabolites that are increased by Sp and that elevate 5-HT in chromaffin cell cultures, suggesting direct metabolic signaling of gut microbes to ECs. Furthermore, elevating luminal concentrations of particular microbial metabolites increases colonic and blood 5-HT in germ-free mice. Altogether, these findings demonstrate that Sp are important modulators of host 5-HT and further highlight a key role for host-microbiota interactions in regulating fundamental 5-HT-related biological processes.

Figures

Figure 1
Figure 1. The Gut Microbiota Modulates Host Peripheral Serotonin Levels
(A) Levels of serum 5-HT. Data are normalized to serum 5-HT in SPF mice. n=8–13. (B) Levels of colon 5-HT relative to total protein. Data are normalized to colon 5-HT relative to total protein in SPF mice. n=8–13. (C) Colonic expression of TPH1 relative to GAPDH. Data are normalized to expression levels in SPF mice. n=4. (D) Colonic expression of SLC6A4 relative to GAPDH. Data are normalized to expression levels in SPF mice. n=4. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.=not statistically significant. SPF=specific pathogen-free (conventionally-colonized), GF=germ-free, CONV.=SPF conventionalized, ABX=antibiotic-treated, VEH=vehicle (water)-treated. See also Figure S1.
Figure 2
Figure 2. Indigenous Spore-forming Bacteria Increase 5-HT Levels in Colon Enterochromaffin Cells
(A) Representative images of colons stained for chromagranin A (CgA) (left), 5-HT (center) and merged (right). Arrows indicate CgA-positive cells that lack 5-HT staining. n=3–7 mice/group. (B) Quantitation of 5-HT+ cell number per area of colonic epithelial tissue. n=3–7 mice/group. (C) Quantitation of CgA+ cell number per area of colonic epithelial tissue. n=3–7 mice/group. (D) Ratio of 5-HT+ cells/CgA+ cells per area of colonic epithelial tissue. n=3–7 mice/group. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. SPF=specific pathogen-free (conventionally-colonized), GF=germ-free, CONV.=SPF conventionalized, ABX=antibiotic-treated, Sp=spore-forming bacteria, PCPA=para-chlorophenylalanine. See also Figure S2.
Figure 3
Figure 3. Indigenous Spore-forming Bacteria Induce Colon 5-HT Biosynthesis and Systemic 5-HT Bioavailability
(A) Levels of serum 5-HT. Data are normalized to serum 5-HT levels in SPF mice. SPF, n=13; GF, n=17; GF+conv.=P21 conventionalization, n=4; SPF+Abx= P42 antibiotic treatment, n=7; B. fragilis monoassociation (BF), n=6; SFB=Segmented Filamentous Bacteria monoassociation, n=4; ASF=Altered Schaedler Flora P21 colonization, n=4; Sp=spore-forming bacteria, P21 colonization, n=4; B. uniformis P21 colonization, n=4; Bd=Bacteroides consortium, n=3. (B) Levels of colon 5-HT relative to total protein. Data are normalized to colon 5-HT relative to total protein in SPF mice. n=5–15. (C) Levels of colon 5-HT relative to total protein after intrarectal treatment with the Tph inhibitor, PCPA, or vehicle. n=4. (D) Colonic expression of TPH1 relative to GAPDH. Data are normalized to expression levels in SPF mice. n=3. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s.=not statistically significant. SPF=specific pathogen-free (conventionally-colonized), GF=germ-free, Sp=spore-forming bacteria, PCPA=para-chlorophenylalanine. See also Figure S3.
Figure 4
Figure 4. Microbiota-Mediated Regulation of Host Serotonin Modulates Gastrointestinal Motility
(A) Total time for transit of orally administered carmine red solution through the GI tract. n=4–8. (B) Defecation rate as measured by number of fecal pellets produced relative to total transit time. n=4–8. (C) Representative images of c-fos and 5HT4 colocalization in the colonic submucosa and muscularis externa. n=4–5 mice/group. (D) Quantitation of total c-fos fluorescence intensity in the colonic submucosa and muscularis externa. n=4–5 mice/group. (E) Quantitation of total 5HT4 fluorescence intensity in the colonic submucosa and muscularis externa. n=4–5 mice/group. (F) Quantitation and representative images of c-fos and calb2 (calretinin) colocalization in the colonic submucosa and muscularis externa. n=5–8 mice/group. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. SPF=specific pathogen-free (conventionally-colonized), GF=germ-free, Sp=spore-forming bacteria, PCPA=para-chlorophenylalanine. See also Figure S4.
Figure 5
Figure 5. Microbiota-Mediated Regulation of Host Serotonin Modulates Hemostasis
(A) Time to cessation of bleeding in response to tail injury. n=7–16. (B) Platelet activation, as measured by percentage of large, high granularity (FSChigh, SSChigh) events after collagen stimulation relative to unstimulated controls. n=3. (C) Representative flow cytometry plots of large, high granularity (FSChigh, SSChigh) activated platelets after collagen stimulation (bottom), as compared to unstimulated controls (top). n=3. (D)–(E) Geometric mean fluorescence intensity of granulophysin (CD63; D), P-selectin (E), JON/A (integrin αIIbβ3; F) expression in collagen-stimulated platelets (left). Representative histograms (right) of event count vs. fluorescence intensity (log scale) for platelets treated with collagen (red line) or vehicle (blue line). n=3. Data for platelet assays are representative of three independent trials with at least three mice in each group. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s.=not statistically significant. SPF=specific pathogen-free (conventionally-colonized), GF=germ-free, Sp=spore-forming bacteria, PCPA=para-chlorophenylalanine. See also Figure S5.
Figure 6
Figure 6. Microbial Metabolites Mediate Effects of the Microbiota on Host Serotonin
(A) Levels of 5-HT released from RIN14B cells after exposure to colonic luminal filtrate from SPF, GF and Sp-colonized mice, or to ionomycin (iono). Data are normalized to 5-HT levels in vehicle-treated controls (hatched gray line at 1). Asterisks directly above bars indicate significance compared to controls; asterisks at the top of the graph denote significance between experimental groups. n=3. (B) Expression of TPH1 relative to GAPDH in RIN14B cells after exposure to colon luminal filtrate from SPF, GF and Sp-colonized mice, or to ionomycin (iono). Data are normalized to gene expression in vehicle-treated controls (hatched gray line at 1). Asterisks directly above bars indicate significance compared to controls, whereas asterisks at the top of the graph denote significance between experimental groups. n=4. (C) Principal components analysis of the fecal metabolome from GF mice colonized with SPF, ASF, Sp, or hSp. n=6. (D) Levels of 5-HT released from RIN14B cells after exposure to metabolites: acetate (1 mM), α-tocopherol (8 uM), arabinose (50 uM), azelate (50 uM), butyrate (100 uM), cholate (75 uM), deoxycholate (25 uM), ferulate (25 uM), GABA (25 uM), glycine (50 uM), N-methyl proline (0.5 uM), oleanolate (50 uM), p-aminobenzoate (1 uM), propionate (100 uM), taurine (50 uM), tyramine (100 uM). Data are normalized to 5-HT levels in vehicle-treated controls (gray line at 1). n=5–19. (E) Expression of TPH1 relative to GAPDH in RIN14B cells after metabolite exposure. Data are normalized to expression in vehicle-treated controls (gray line at 1). n=3–4. (F) Levels of 5-HT in colons (left) and serum (center) of GF mice at 30 min after intrarectal injection of deoxycholate (125 mg/kg) or vehicle. Expression of TPH1 relative to GAPDH (right) at 1 hr post injection. n=3–8. (G) Phylogenetic tree displaying key Sp. (M) and hSp. (H) operational taxonomic units (OTUs) relative to reference Clostridium species with reported 7α-dehydroxylation activity (red circles). Relative abundances are indicated in parentheses. n=3. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s.=not statistically significant. SPF=specific pathogen-free (conventionally-colonized), GF=germ-free, Sp=spore-forming bacteria, iono=15uM ionomycin, ASF=Altered Schaedler Flora, hSp=humanderived spore-forming bacteria. See also Figures S6 and S7.

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