HHS Public Access Author manuscript Cell, Author manuscript; available in PMC 2016 April 09 Published in final edited form as Cell2015Aprl9161(2):264-276.doi:10.1016/cl2015.02047 Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis Jessica M Yano, Kristie Yu, Gregory P. Donaldson, Gauri G. Shastri, Phoebe Ann' Liang Ma2, Cathryn R Nagler, Rustem F Ismagilov, Sarkis K Mazmanian, and Elaine Y. 1Division of Biology Biological Engineering, California Institute of Technology, Pasadena, CA 91125.USA E9乙 Division of Chemistry and Chemical Engineering California Institute of Technology, Pasadena CA 91125 USA 3Department of Pathology and Department of Medicine, The Unit of Chicago, Chicago, IL 60637.USA SUMMARY The gastrointestinal(GI) tract contains much of the bodys serotonin (5-hydroxytryptamine, 5- IT), 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 Gl 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 0 2015 Published by Elsevier Inc. to ehsiaoacaltec Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of he resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain 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 he 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
Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis Jessica M. Yano1, Kristie Yu1, Gregory P. Donaldson1, Gauri G. Shastri1, Phoebe Ann1, Liang Ma2, Cathryn R. Nagler3, Rustem F. Ismagilov2, Sarkis K. Mazmanian1, and Elaine Y. Hsiao1,* 1Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA 2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA 3Department 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 sporeforming 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. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 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 Author manuscript Cell. Author manuscript; available in PMC 2016 April 09. Published in final edited form as: Cell. 2015 April 9; 161(2): 264–276. doi:10.1016/j.cell.2015.02.047. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Yano et al and other organ systems. More than 90% of the body's 5-HT is synthesized in the gut, wher 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) nd 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 ns of ls of gut 5-HT wo different isoenzymes of tryptophan hydroxylase(Tph), TphI 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 egulate gl motility and hemostasis in the host, suggesting that targeting the microbiota can Cell. Author manuscript; available in PMC 2016 April 09
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 nonneuronal 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 Yano et al. Page 2 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Yano et al 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 IA)and plasma(Figure SIA)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 Gl tract, we find that GF mice exhibit significantly decreased levels of colonic and fecal 5-HT compared to SPF controls(Figures IB and SIA, and Table S1). This deficit in 5-HT is observed broadly across the distal medial and proximal colon(Figure SID), but not in the small intestine(Figures SIA, S2A and S2B), suggesting a specific role for the microbiota in regulating colonic 5-HT 9 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 TPHl(Figures IC and SID; 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 SIC) GF mice also display elevated colonic expression of the 5-HT transporter SLC6A4 (Figures ID and SIE; 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 clonic expression of neural-specific isoforms of 5-HT enzymes(Figure SIF), 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, IB and SIA, 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 TPHI 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 SIG, SIH and SIn). Collectively, these data support the notion that the microbiota promotes 5-HT biosynthesis by elevating TPHl 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 Cell. Author manuscript; available in PMC 2016 April 09
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 Yano et al. Page 3 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Yano et al crobiota at birth(postnatal day(P)O), weaningP21), 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 olonization. Colonic expression of TPHI and SLC6A4 is similarly corrected by postnatal conventionalization of GF mice( Figures IC and lD), with more substantial changes from PO 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 9 gut microbiota in SPF mice via bi-daily antibiotic treatment beginning on PO, P2l 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 TPHI and SlC6A4 expression(Figures I and 2). Interestingly, PO and P21 antibiotic treatment also induces GF related deficits in colonic 5-HT, but the effects on serum 5-HT are more pronounced when dministered at P42, compared to PO 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 PO 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 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 esponsible for affecting host 5-HT pathways. Mice monocolonized with Bacteroides fragilis or Segmented Filamentous Bacteria(SFB) display deficits in serum 5-HT that are omparable 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, GF mice colonized at P42 with indigenous spore-forming microbes from the (Sp), known to be dominated by Clostridial species(Atarashi et al Stefka et al 2014)(Figure S7 and Table $4), exhibit complete restoration of serum and colon 5-HTto levels observed in SPF mice(Figures 3A and 3B). Consistent with this, Sp colonization of Cell. Author manuscript; available in PMC 2016 April 09
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 microbiotamediated 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 GFrelated 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 Yano et al. Page 4 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Yano et al GF mice increases 5-HT staining colocalized to CgA+ ECs( Figure 2), elevates host coloni TPHI 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 Ragl 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 ith 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- c9 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 TPHI 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 SIc6a4-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 colonic ECs from SLC6A4 +/-and-/-mice(Figure $4C). Similar effects of antibiotic treatment and Sp colonization are seen for serum 5-HT in SLC6A4+/-mice, whereas SLC6A4-1-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 Tphl-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 gl 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 Cell. Author manuscript; available in PMC 2016 April 09
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 parachlorophenylalanine (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 microbiotamediated 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 Spinduced 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 Yano et al. Page 5 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript