Indigenous bacteria from the gut microbiota regulate host
serotonin biosynthesis
Jessica M. Yano
1
,
Kristie Yu
1
,
Gregory P. Donaldson
1
,
Gauri G. Shastri
1
,
Phoebe Ann
1
,
Liang Ma
2
,
Cathryn R. Nagler
3
,
Rustem F. Ismagilov
2
,
Sarkis K. Mazmanian
1
, and
Elaine Y.
Hsiao
1,*
1
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA
91125, USA
2
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
CA 91125, USA
3
Department of Pathology and Department of Medicine, The University of Chicago, Chicago, IL
60637, USA
SUMMARY
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.
INTRODUCTION
In addition to its role as a brain neurotransmitter, the monoamine serotonin (5-
hydroxytryptamine, 5-HT) is an important regulatory factor in the gastrointestinal (GI) tract
© 2015 Published by Elsevier Inc.
*
Correspondence to: ehsiao@caltech.edu.
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AUTHORS CONTRIBUTIONS
J.M.Y., K.Y., G.P.D., G.G.S., P.A., L.M. and E.Y.H. performed the experiments and analyzed the data, J.M.Y. and E.Y.H. designed
the study, C.R.N., R.F.I and S.K.M. provided novel reagents, R.F.I. and S.K.M. provided valuable support and contributed equally,
J.M.Y. and E.Y.H. wrote the manuscript. All authors discussed the results and commented on the manuscript.
HHS Public Access
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Cell
. 2015 April 9; 161(2): 264–276. doi:10.1016/j.cell.2015.02.047.
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and other organ systems. More than 90% of the body’s 5-HT is synthesized in the gut, where
5-HT activates as many as 14 different 5-HT receptor subtypes (
Gershon and Tack, 2007
)
located on enterocytes (
Hoffman et al., 2012
), enteric neurons (
Mawe and Hoffman, 2013
)
and immune cells (
Baganz and Blakely, 2013
). In addition, circulating platelets sequester 5-
HT from the GI tract, releasing it to promote hemostasis and distributing it to various body
sites (
Amireault et al., 2013
). As such, gut-derived 5-HT regulates diverse functions,
including enteric motor and secretory reflexes (
Gershon and Tack, 2007
), platelet
aggregation (
Mercado et al., 2013
), immune responses (
Baganz and Blakely, 2013
) and bone
development (
Chabbi-Achengli et al., 2012
;
Yadav et al., 2008
) and cardiac function (
Cote
et al., 2003
). Furthermore, dysregulation of peripheral 5-HT is implicated in the
pathogenesis of several diseases, including irritable bowel syndrome (IBS) (
Stasi et al.,
2014
), cardiovascular disease (
Ramage and Villalon, 2008
) and osteoporosis (
Ducy and
Karsenty, 2010
).
The molecular mechanisms controlling the metabolism of gut 5-HT remain unclear. In the
GI tract, 5-HT is synthesized by specialized endocrine cells, called enterochromaffin cells
(ECs), as well as mucosal mast cells and myenteric neurons (
Gershon and Tack, 2007
), but
the functions of these different pools of gut 5-HT are incompletely understood. In addition,
two different isoenzymes of tryptophan hydroxylase (Tph), Tph1 and Tph2, mediate non-
neuronal vs. neuronal 5-HT biosynthesis (
Walther et al., 2003
), but little is known regarding
the endogenous signals that regulate Tph expression and activity.
Mammals are colonized by a vast and diverse collection of microbes that critically
influences health and disease. Recent studies highlight a role for the microbiota in regulating
blood 5-HT levels, wherein serum concentrations of 5-HT are substantially reduced in mice
reared in the absence of microbial colonization (germ-free, GF), compared to
conventionally-colonized (specific pathogen-free, SPF) controls (
Sjogren et al., 2012
;
Wikoff et al., 2009
). In addition, intestinal ECs are morphologically larger in GF vs. SPF
rats (
Uribe et al., 1994
), which suggests that microbes could impact the development and/or
function of 5-HT-producing cells. Interestingly, some species of bacteria grown in culture
can produce 5-HT (
Tsavkelova et al., 2006
), raising the question of whether indigenous
members of the microbiota contribute to host 5-HT levels through
de novo
synthesis. Based
on this emerging link between the microbiota and serum 5-HT concentrations, we aimed to
determine how pathways of 5-HT metabolism are affected by the gut microbiota, to identify
specific microbial communities and factors involved in conferring serotonergic effects and
to evaluate how microbial modulation of peripheral 5-HT impacts host physiology.
We show herein that the microbiota promotes 5-HT biosynthesis from colonic ECs in a
postnatally inducible and reversible manner. Spore-forming microbes (Sp) from the healthy
mouse and human microbiota sufficiently mediate microbial effects on serum, colon and
fecal 5-HT levels. We further explore potential host-microbial interactions that regulate
peripheral 5-HT by surveying microbial influences on the fecal metabolome. We find that
particular microbial metabolites are elevated by Sp and likely signal directly to colonic ECs
to promote 5-HT biosynthesis. Importantly, microbiota-mediated changes in colonic 5-HT
regulate GI motility and hemostasis in the host, suggesting that targeting the microbiota can
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serve as a tractable approach for modulating peripheral 5-HT bioavailability and treating 5-
HT-related disease symptoms.
RESULTS
The Gut Microbiota Modulates Host Peripheral Serotonin Levels
Adult GF mice display deficient serum (
Sjogren et al., 2012
;
Wikoff et al., 2009
) (Figure
1A) and plasma (Figure S1A) 5-HT concentrations compared to SPF controls, but the
cellular sources of this disruption are undefined. Consistent with the understanding that
much of the body’s 5-HT derives from the GI tract, we find that GF mice exhibit
significantly decreased levels of colonic and fecal 5-HT compared to SPF controls (Figures
1B and S1A, and Table S1). This deficit in 5-HT is observed broadly across the distal,
medial and proximal colon (Figure S1D), but not in the small intestine (Figures S1A, S2A
and S2B), suggesting a specific role for the microbiota in regulating colonic 5-HT.
Decreased levels of 5-HT are localized to colonic chromogranin A− positive (CgA+)
enterochromaffin cells (ECs) (Figure 2), and not to small intestinal ECs (Figures S2A and
S2B). Low 5-HT signal is seen in both GF and SPF colonic mast cells and enteric neurons
(Figure 2A), which are minor producers of 5-HT (
Gershon and Tack, 2007
). There is no
difference between adult GF and SPF mice in the abundance of CgA+ enteroendocrine cells
(EECs) (Figure 2C), suggesting that decreases in colon 5-HT result from abnormal 5-HT
metabolism rather than impaired development of EECs.
To identify the specific steps of 5-HT metabolism that are affected by the microbiota, key
intermediates of the 5-HT pathway were assessed in colons from GF vs. SPF mice. We find
that GF colons exhibit decreased expression of
TPH1
(Figures 1C and S1D;
Sjogren et al.,
2012
), the rate-limiting enzyme for 5-HT biosynthesis in ECs, but no difference in
expression of enzymes involved in 5-HT packaging, release and catabolism (Figure S1C).
GF mice also display elevated colonic expression of the 5-HT transporter
SLC6A4
(Figures
1D and S1E;
Sjogren et al., 2012
), synthesized broadly by enterocytes to enable 5-HT
uptake (
Wade et al., 1996
). This could reflect a compensatory response to deficient 5-HT
synthesis by host ECs, based on the finding that chemical Tph inhibition modulates
SLC6A4
expression (Figures S2C and S2D). There is no difference between GF and SPF mice in
colonic expression of neural-specific isoforms of 5-HT enzymes (Figure S1F), consistent
with data showing no apparent difference in 5-HT-specific staining in enteric neurons
(Figure 2). Despite deficient levels of colon, fecal and serum 5-HT (Figures 1A, 1B and
S1A, and Table S1), GF mice exhibit significantly increased levels of the Tph substrate,
tryptophan (Trp), in both feces (Table S1) and serum (
Sjogren et al., 2012
;
Wikoff et al.,
2009
), suggesting that primary disruptions in host
TPH1
expression result in Trp
accumulation. Oral supplementation of GF mice with the Tph product, 5-hydroxytryptophan
(5-HTP), sufficiently ameliorates deficits in colon and serum 5-HT, whereas
supplementation with the Tph substrate Trp has no restorative effect (Figures S1G, S1H and
S1I). Collectively, these data support the notion that the microbiota promotes 5-HT
biosynthesis by elevating
TPH1
expression in colonic ECs.
To confirm that deficient 5-HT levels in GF mice are microbiota-dependent, and further
determine whether effects are age-dependent, GF mice were conventionalized with an SPF
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microbiota at birth (postnatal day (P) 0), weaning (P21), or early adulthood (P42) and then
evaluated at P56 for levels of 5-HT and expression of 5-HT-related genes. GF mice
conventionalized at each age with an SPF microbiota exhibit restored serum (Figure 1A) and
colon (Figure 1B) 5-HT levels, with more pronounced effects seen at earlier ages of
colonization. Colonic expression of
TPH1
and
SLC6A4
is similarly corrected by postnatal
conventionalization of GF mice (Figures 1C and 1D), with more substantial changes from
P0 conventionalization. Increases in 5-HT are localized to colonic ECs (Figure 2). These
findings indicate that postnatal reconstitution of the gut microbiota can correct the 5-HT
deficiency seen in GF mice and further suggest that gut microbes exert a continuous effect
on 5-HT synthesis by modulating EC function. Overall, we demonstrate that microbiota-
mediated elevation of host 5-HT is postnatally inducible, persistent from the time of
conventionalization and not dependent on the timing of host development.
To assess the reversibility of microbial effects on host 5-HT metabolism, we depleted the
gut microbiota in SPF mice via bi-daily antibiotic treatment beginning on P0, P21 or P42
and until P56. Treatment of P42 SPF mice with a cocktail of ampicillin, vancomycin,
neomycin and metronidazole (Reikvam et al., 2011) sufficiently recapitulates GF-associated
deficits in serum and colon 5-HT and alterations in host colonic
TPH1
and
SLC6A4
expression (Figures 1 and 2). Interestingly, P0 and P21 antibiotic treatment also induces GF-
related deficits in colonic 5-HT, but the effects on serum 5-HT are more pronounced when
administered at P42, compared to P0 and P21 (Figure 1), suggesting potential confounding
effects of early life or prolonged antibiotic treatment on microbiota-mediated modulation of
peripheral 5-HT. Antibiotics can elicit several direct effects on host cells (
Shimizu et al.,
2003
;
Westphal et al., 1994
), which may underlie differences between P0 treatment and GF
status. That P42 antibiotic treatment of SPF mice results in 5-HT phenotypes analogous to
those seen in GF mice demonstrates that microbiota effects on host 5-HT can be abrogated
postnatally and further supports the plasticity of 5-HT modulation by indigenous gut
microbes. Altogether, these data indicate that the gut microbiota plays a key role in raising
levels of colon and serum 5-HT, by promoting 5-HT in colonic ECs in an inducible and
reversible manner.
Indigenous Spore-Forming Microbes Promote Host Serotonin Biosynthesis
In light of our finding that 5-HT levels are decreased in colons but not small intestines of GF
mice compared to SPF controls, we hypothesized that specific subsets of gut microbes are
responsible for affecting host 5-HT pathways. Mice monocolonized with
Bacteroides
fragilis
or Segmented Filamentous Bacteria (SFB) display deficits in serum 5-HT that are
comparable to those seen in GF mice (Figure 3A). Moreover, postnatal colonization (P42)
with
Bacteroides uniformis
, altered Schaedler flora (ASF), an eight-microbe consortium
known to correct gross intestinal pathology in GF mice (
Dewhirst et al., 1999
), or with
cultured
Bacteroides spp
. from the SPF mouse microbiota, has no significant effect on the 5-
HT deficiency seen in GF mice (Figures 3A and 3B). Interestingly, however, GF mice
colonized at P42 with indigenous spore-forming microbes from the mouse SPF microbiota
(Sp), known to be dominated by Clostridial species (
Atarashi et al., 2013
;
Stefka et al.,
2014
) (Figure S7 and Table S4), exhibit complete restoration of serum and colon 5-HT to
levels observed in SPF mice (Figures 3A and 3B). Consistent with this, Sp colonization of
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GF mice increases 5-HT staining colocalized to CgA+ ECs (Figure 2), elevates host colonic
TPH1
expression (Figure 3D) and decreases
SLC6A4
expression (Figure 3E) toward levels
seen in SPF mice. Improvements in serum 5-HT are observed within 2 days after inoculation
of GF mice with Sp (Figure S2E), and do not correlate with amelioration of abnormal cecal
weight (Figure S2F). Importantly, Sp also elevates colonic 5-HT in Rag1 knockout mice
(Figure S2G), which lack adaptive immune cells, indicating that the effects of Sp on gut 5-
HT are not dependent on Sp-mediated regulatory T cell induction (
Stefka et al., 2014
).
Notably, the 5-HT-promoting effects of Sp are recapitulated by colonization of GF mice
with spore-forming microbes from the healthy human colonic microbiota (hSp) (Figure S3),
suggesting that the serotonergic function of this community is conserved across mice and
humans.
To determine whether the effects of Sp on host 5-HT depend on colonic Tph activity, we
colonized GF mice with Sp on P42 and then administered the Tph inhibitor para-
chlorophenylalanine (PCPA) intrarectally twice daily for 3 days prior to 5-HT assessments
on P56 (Liu et al., 2008). Intrarectal injection of PCPA sufficiently blocks the ability of Sp
to elevate colon and serum 5-HT levels (Figures 3C and S2C), as well as Sp-mediated
increases in 5-HT staining in ECs (Figure 2). Similar effects of PCPA treatment on blocking
increases in colon 5-HT, serum 5-HT and 5-HT staining in colonic ECs are seen in GF mice
colonized with hSp (Figure S3). Interestingly, inhibiting Tph activity with PCPA results in a
compensatory increase in colonic
TPH1
and decrease in
SLC6A4
(Figures 3D and S2D)
expression in Sp-colonized mice, supporting the notion that microbiota-dependent changes
in 5-HT transporter levels occur as a secondary response to Tph modulation.
To further evaluate whether changes in
SLC6A4
expression are necessary for microbiota-
mediated alterations in peripheral 5-HT, we tested the effects of microbiota manipulations
on colon and serum 5-HT in
SLC6A4
heterozygous (+/−) and complete (−/−) knockout (KO)
mice. Depleting the microbiota via P42-P56 antibiotic treatment (Reikvam et al., 2011) of
SPF
SLC6A4
+/− and −/− mice effectively decreases colonic 5-HT levels (Figures S4A and
S4B), indicating that the microbiota is required for promoting gut 5-HT in Slc6a4-deficient
mice. Colonizing antibiotic-treated
SLC6A4
+/− and −/− mice with Sp raises colon 5-HT to
levels seen in SPF
SLC6A4
+/− and −/− mice (Figure S4A), demonstrating that Slc6a4 is not
required for conferring the effects of Sp on gut 5-HT. Antibiotic-induced decreases and Sp-
induced increases in colon 5-HT levels can be attributed to modulation of 5-HT content in
colonic ECs from
SLC6A4
+/− and −/− mice (Figure S4C). Similar effects of antibiotic
treatment and Sp colonization are seen for serum 5-HT in
SLC6A4
+/− mice, whereas
SLC6A4
−/− mice exhibit low to undetectable levels of serum 5-HT, highlighting the
dependence of platelets on Slc6a4-mediated 5-HT uptake (Figure S4B). Taken together,
these data support a role for Sp in promoting Tph1-mediated 5-HT biosynthesis by colonic
ECs, regulating both colon and serum levels of 5-HT.
Microbiota-Mediated Regulation of Host Serotonin Modulates Gastrointestinal Motility
Intestinal 5-HT plays an important role in stimulating the enteric nervous system and GI
function (
Gershon and Tack, 2007
). To determine whether microbiota-dependent
modulation of colonic 5-HT impacts GI motility, we colonized P42 GF mice with Sp and
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then tested for GI transit and colonic neuronal activation at P56. Sp colonization ameliorates
GF-associated abnormalities in GI motility, significantly decreasing total transit time and
increasing the rate of fecal output in a Tph-dependent manner (Figures 4A and 4B). Similar
effects are seen in
SLC6A4
+/− and −/− mice, where Sp colonization of antibiotic-treated
mice restores GI transit time toward levels seen in SPF
SLC6A4
+/− and −/− controls
(Figure S4E).
Consistent with deficits in GI motility, steady-state activation of 5-HT receptor subtype 4
(5HT4)-expressing neurons in the colonic submucosa and muscularis externa is decreased in
GF mice compared to SPF controls, as measured by colocalized expression of 5HT4 with
the immediate early gene, c-fos (Figure 4C, 4D and 4E). Colonization of GF mice with Sp
increases 5HT4+ c-fos+ staining to levels seen in SPF mice, and this effect is dependent on
colonic Tph activity (Figures 4C, 4D and 4E), which aligns well with the understanding that
Sp-induced elevations in colonic 5-HT promote GI motility by activation of 5HT4+ enteric
neurons (
Mawe and Hoffman, 2013
). In addition, colonic activation of intrinsic afferent
primary neurons (IPANs) of the myenteric plexus is decreased in GF mice (
McVey Neufeld
et al., 2013
) and improved by colonization with Sp, as measured by colocalization of c-fos
and the IPAN marker, calretinin (Calb2) (Figure 4F). Inhibiting Tph activity with PCPA
decreases IPAN activation in Sp-colonized mice, suggesting that some IPAN responses to
Sp depend on host 5-HT synthesis (Figures 4F). Altogether, these findings indicate that Sp-
mediated increases in colonic 5-HT biosynthesis are important for gut sensorimotor
function.
Microbiota-Mediated Regulation of Host Serotonin Modulates Platelet Function
Platelets uptake gut-derived 5-HT and release it at sites of vessel injury to promote blood
coagulation. To determine if microbiota-dependent modulation of colon (Figures 1 and 3)
and plasma (Figure S1A) 5-HT impacts platelet function, we colonized P42 mice with Sp
and then examined blood clotting, platelet activation and platelet aggregation at P56. In a
tail bleed assay (Liu et al., 2012), GF mice exhibit trending increases in time to cessation of
bleeding compared to SPF mice, suggesting impaired blood coagulation (Figure 5A).
Colonization of GF mice with Sp ameliorates abnormalities in bleeding time to levels seen
in SPF controls, and this effect is attenuated by intrarectal administration of PCPA (Figure
5A), indicating that Sp-mediated improvements in coagulation may be dependent on colonic
Tph activity. Notably, the impact of acute colonic PCPA treatment on reducing 5-HT
content and 5-HT-related functions in platelets may be tempered by the fact that mouse
platelets have a lifespan of ~4 days (
Odell and Mc, 1961
). There were no significant
differences between treatment groups in total platelet counts (Figure S5A).
In light of inherent limitations of the tail bleed assay (Liu et al., 2012), we focused
subsequent experiments particularly on platelet activity. Platelets isolated from GF mice
display decreased activation in response to
in vitro
type I fibrillar collagen stimulation, as
measured by reduced surface expression of the activation markers granulophysin (CD63), P-
selectin and JON/A (integrin
α
IIb
β
3) (Figures 5D, 5E and 5F) (
Ziu et al., 2012
). Sp
colonization of GF mice leads to partial restoration in the expression of platelet activation
markers, and this effect depends on colonic Tph activity (Figures 5D, 5E and 5F). Moreover,
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platelets isolated from GF mice exhibit impaired aggregation in response to
in vitro
collagen
stimulation, as measured by decreased levels of high granularity, high mass aggregates
detected by both flow cytometry (De Cuyper et al., 2013; Nieswandt et al., 2004) (Figures
5B 5C, S5C an S5D) and imaging (Figure S5B). Colonization of GF mice with Sp restores
levels of platelet aggregation to those seen in SPF mice. These effects of Sp on correcting
impaired platelet aggregation are attenuated by colonic PCPA injection, indicating
dependence on Tph activity. Overall, these findings suggest that Sp-mediated elevations in
colonic 5-HT, and thus platelet 5-HT, promote platelet activation and aggregation relevant
to hemostasis.
Microbial Metabolites Mediate Effects of the Microbiota on Host Serotonin
In light of the important role for Sp in regulating 5-HT-related intestinal and platelet
function, we aimed to identify specific microbial factors responsible for conferring the
serotonergic effects of Sp. Based on our finding that Sp elevates 5-HT particularly in colonic
ECs (Figure 2), we hypothesized that Sp promotes levels of a soluble factor that signals
directly to ECs to modulate
TPH1
expression and 5-HT biosynthesis. To test this, we
prepared filtrates of total colonic luminal contents from Sp-colonized mice and controls, and
evaluated their effects on levels of 5-HT in RIN14B chromaffin cell cultures (Nozawa et al.,
2009). Relative to vehicle-treated controls, there is no significant effect of filtered colonic
luminal contents from GF mice on levels of 5-HT released or
TPH1
expressed from RIN14B
cells (Figures 6A and 6B). Filtered colonic luminal contents from SPF and Sp-colonized
mice sufficiently induce 5-HT from RIN14B cells (Figure 6A), to levels comparable to those
elicited by the calcium ionophore, ionomycin, as a positive control.
TPH1
expression is also
elevated in chromaffin cells exposed to SPF and Sp luminal filtrates, suggesting increased 5-
HT synthesis. This is in contrast to ionomycin, which stimulates 5-HT release, but has no
effect on
TPH1
expression, from RIN14B cells. Importantly, these findings suggest that
microbiota-mediated increases in gut 5-HT are conferred via direct signaling of a soluble,
Sp-modulated factor to colonic ECs.
We utilized metabolomic profiling to identify candidate Sp-dependent, 5-HT-inducing
molecules in feces from adult mice. Sp colonization of GF mice leads to statistically
significant alterations in 75% of the 416 metabolites detected, of which 76% are elevated
and 24% are reduced, relative to vehicle-treated GF controls (Tables S1 and S2). Similar
changes are seen with hSp colonization, leading to co-clustering of Sp and hSp samples by
principal components analysis (PCA) (Figure 6C). ASF colonization has a mild effect,
significantly modulating 50% of metabolites detected (66% increased, 36% decreased)
(Table S2), and forming a distinct but proximal cluster to GF controls by PCA (Figure 6C).
Postnatal conventionalization of GF mice with an SPF microbiota alters 66% of all
metabolites detected (59% increased, 41% decreased) (Table S2), and produces substantial
changes in the metabolome that are distinguishable from the effects of Sp, hSp and ASF
along PC2 (Figure 6C). Notably, Sp, hSp and SPF colonization results in similar shifts along
PC1, compared to vehicle and ASF-treated controls, suggesting common metabolic
alterations among communities that similarly elevate peripheral 5-HT levels. Metabolomics
profiling confirms that fecal 5-HT is commonly upregulated in the Sp, hSp and SPF fecal
metabolome, and comparatively low in ASF and GF samples (Table S1). Simple linear
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regression reveals 83 metabolites that co-vary with 5-HT (r
2
≥ 0.25), 47 of which correlate
positively and 36 of which correlate negatively with 5-HT levels (Table S3 and Figure S6A).
To determine whether specific metabolites mediate the effects of Sp on 5-HT, we tested a
subset of biochemicals that were commonly upregulated by Sp, hSp and SPF, and that
positively correlated with 5-HT levels (Table S3 and Figure S6A), for their ability to induce
5-HT
in vitro
and
in vivo
. We also tested the short chain fatty acids, acetate, butyrate and
propionate, which were previously shown to be produced by Sp (
Atarashi et al., 2013
) and
to stimulate 5-HT release from ECs (
Fukumoto et al., 2003
). Of 16 metabolites examined,
α
-tocopherol, butyrate, cholate, deoxycholate, p-aminobenzoate (PABA), propionate and
tyramine elevate 5-HT in RIN14B chromaffin cell cultures (Figure 6D). Elevations in 5-HT
correspond to increases in
TPH1
expression from RIN14B cells (Figure 6E), suggesting that
particular metabolites induced by Sp enhance 5-HT biosynthesis by ECs. We further tested
for sufficiency to induce 5-HT
in vivo
. Notably, raising luminal concentrations of
deoxycholate in colons of GF mice to levels seen in SPF mice (
Sayin et al., 2013
)
sufficiently increases colon and serum 5-HT compared to vehicle-injected controls (Figures
6F and S6B). This restoration of peripheral 5-HT correlates with elevations in colonic
TPH1
expression (Figure 6F). Increases in colon and serum 5-HT are also seen with injection of
α
-
tocopherol, PABA and tyramine into colons of GF mice (Figures S6B and S6C). Consistent
with
in vitro
RIN14B data, oleanolate has no statistically significant effect on elevating
colon or serum 5-HT in GF mice (Figures S6B and S6C). Importantly, the effects of a single
rectal injection of deoxycholate or
α
-tocopherol on raising colon 5-HT levels in GF mice are
weak and transient, peaking within 1 hour of injection (Figure S6C). Consistent with this,
there is no significant effect of acute colonic metabolite injection on GI transit time (Figure
S6D), and there is only a trending improvement on platelet activation (Figure S6E). Our
finding that Sp colonization leads to lasting increases in colon and blood 5-HT levels
(Figure 3), and long-term changes in the fecal metabolome (Figure 6C and Tables S1 and
S2), suggests that Sp colonization results in persistent elevations of 5-HT-modulating
luminal metabolites. Future studies on whether chronic, colon-restricted increases in Sp-
regulated metabolites sufficiently correct GI motility and platelet function in GF mice, and
whether this occurs in a 5-HT-dependent manner, are warranted. In addition, we
demonstrate that select concentrations of Sp-associated metabolites sufficiently promote 5-
HT
in vitro
and
in vivo
, but whether the metabolites are necessary for mediating the
serotonergic effects of Sp is unclear. Overall, these data reveal that indigenous spore-
forming microbes promote 5-HT biosynthesis from colonic ECs, modulating 5-HT
concentrations in both colon and blood. Furthermore, we identify select microbial
metabolites that confer the serotonergic effects of indigenous spore-forming microbes, likely
by signaling directly to colonic ECs to promote Tph1 expression and 5-HT biosynthesis.
DISCUSSION
The GI tract is an important site for 5-HT biosynthesis, but the regulatory mechanisms
underlying the metabolism of gut-derived 5-HT are incompletely understood. Here we
demonstrate that the gut microbiota plays a key role in promoting levels of colon and blood
5-HT, largely by elevating synthesis by host ECs. This host-microbiota interaction
contributes to a growing appreciation that the microbiota regulates many aspects of GI
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physiology by signaling to host cells. Whether particular members of the microbiota
contribute 5-HT by
de novo
synthesis remains unclear. Some bacteria, including
Corynebacterium
spp.,
Streptococcus
spp. and
Escherichia coli
, are reported to synthesize 5-
HT in culture (
Roshchina, 2010
), but this is believed to occur independently of Tph, by
decarboxylation of tryptophan to tryptamine (
Williams et al., 2014
), as seen in plants
(
Oleskin et al., 1998
). Our finding that colonic PCPA administration blocks the ability of the
microbiota to promote colonic and blood 5-HT (Figures 3C and 3D) suggests that gut
microbes require host Tph activity to upregulate peripheral 5-HT. Furthermore, SPF Tph1
KO mice lack >90% of intestinal and blood 5-HT levels (
Savelieva et al., 2008
), indicating
that <10% of peripheral 5-HT is contributed directly by microbial synthesis or by Tph2-
mediated biosynthesis in these mice. We find that the microbiota regulates relatively high
levels of peripheral 5-HT, 64% of colonic (Figure 1) and 49% of serum concentrations
(Figure 1;
Sjogren et al., 2012
;
Wikoff et al., 2009
), further supporting the notion that the
microbiota modulates 5-HT metabolism primarily by affecting host colonic ECs. Consistent
with the understanding that ECs secrete low levels of 5-HT into the lumen, fecal
concentrations of 5-HT are also significantly increased by the microbiota. Interestingly, 5-
HT is reported to stimulate the growth of
Enterococcus faecalis, E. coli
and
Rhodospirillum
rubrum
in culture (
Oleskin et al., 1998
;
Tsavkelova et al., 2006
). In addition, 5-HT is a
structural analogue of auxins found in
E. faecalis, R. rubrum
and
Staphylococcus aureus
,
among other bacteria. Whether particular members of the microbiota alter host 5-HT
biosynthesis to, in turn, support colonization, growth or resilience of particular gut microbes
is an interesting question for future study.
We demonstrate that indigenous spore-forming microbes from colons of SPF mice (Sp) and
from a healthy human colon (hSp) sufficiently mediate microbiota effects on colonic and
blood 5-HT. While we show that
B. fragilis, B. uniformis
, SFB, ASF and a consortium of
Bacteroides
species cultured from mice, including
B. thetaiotaomicron, B. acidifaciens and
B. vulgatus
, have no effect on host peripheral 5-HT (Figure 3), whether other non-Sp
microbial species or communities are capable of modulating colonic and serum 5-HT
remains unclear. Interestingly, Sp and hSp are known to promote regulatory T cell levels in
the colons, but not small intestines, of GF and SPF mice (
Atarashi et al., 2013
). This
regional specificity is also seen with microbiota-induced 5-HT biosynthesis, which occurs in
colonic, but not small intestinal, ECs (Figures S1A, S2A and S2B). We find that Sp elevates
colon 5-HT levels even in Rag1 KO mice (Figure S2G), indicating that the serotonergic
effects of Sp are not dependent on T and B cells. Whether 5-HT modulation contributes to
the immunosuppressive effects of Sp, however, is unclear. In light of increasing evidence
that innate and adaptive immune cells express a variety of 5-HT receptors (
Baganz and
Blakely, 2013
), future studies examining whether Sp-mediated increases in peripheral 5-HT
levels impact cellular immune responses will be of interest.
Consistent with our finding that the microbiota modulates colon and serum 5-HT via
interactions with host colonic ECs, we find that particular fecal metabolites are similarly
elevated by SPF, Sp and hSp microbiota, and sufficiently promote 5-HT in chromaffin cell
cultures and
in vivo
(Figure 6 and Table S3). Deoxycholate is a secondary bile acid,
produced by microbial biotransformation of cholate. Notably, deoxycholate is reported to
Yano et al.
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