Yano et al Page 6 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 ffects are seen in SLC6A4+/-and--mice, where Sp colonization of antibiotic-treated mice restores Gi transit time toward levels seen in SPF SLC644 +/and -/-controls Consistent with deficits in GI motility, steady-state activation of 5-HT receptor subtype 4 (S5HT4-expressing neurons in the colonic submucosa and muscularis externa is decreased 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-fost 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 Gl motility by activation of 5HT4+ enteric 9 neurons(Mawe and Hoffman, 2013). In addition, colonic activation of intrinsic afferent primary neurons(IPANs)of the myenteric plexus is decreased in GF mice(Mc Vey 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 S 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 I and 3) and plasma(Figure SlA)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- (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, Cell. Author manuscript; available in PMC 2016 April 09
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 Spmediated 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), Pselectin and JON/A (integrin αIIbβ3) (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, Yano et al. Page 6 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Yano et al 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 c9 ECs(Figure 2), we hypothesized that Sp promotes levels of a soluble factor that signal directly to ECs to modulate TPHI 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 uminal contents from GF mice on levels of 5-HT released or TPHl 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. TPHI 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 ffect on TPHl expression, from RIN14B cells Importantly, these findings suggest that microbiota-mediated increases in gut 5-HT are conferred via direct signaling of a soluble, dulated 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 ignificant alterations in 75% of the 416 metabolites detected, of which 76% are elevated and 24% are reduced, relative to vehicle-treated gF controls (Tables SI 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 PCl, 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 Cell. Author manuscript; available in PMC 2016 April 09
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 Yano et al. Page 7 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Yano et al regression reveals 83 metabolites that co-vary with 5-HT([>0.25),47 which correlate positively and 36 of which correlate negatively with 5-HT levels (Table $3 and Figure S6A) To determine whether specific metabolites mediate the effects of Sp on 5-HT, we tested a abset 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, a-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 TPHI 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 9 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 TPHI expression(Figure 6F). Increases in colon and serum 5-HT are also seen with injection of a tocopherol, PABA and tyramine into colons of GF mice(Figures S6B and S6C). Consister ith 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 a-tocopherol on raising colon 5-HT levels in GF mice are nin 1 hour of there S6D), and there is only a trending improvement on platelet activation( Figure S6E) O 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 SI and S2), suggests that Sp colonization results in persistent elevations of 5-HT-modulating urinal metabolites. Future studies on whether chronic, colon-restricted increases in Sp- regulated metabolites sufficiently correct Gl 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 oncentrations 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 TphI 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 HT, largely by elevating synthesis by host ECs. This host-microbiota interaction ontributes to a growing appreciation that the microbiota regulates many aspects of gl Cell. Author manuscript; available in PMC 2016 April 09
regression reveals 83 metabolites that co-vary with 5-HT (r2 ≥ 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 Spregulated 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 sporeforming 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 Yano et al. Page 8 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Yano et al 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 th 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 Tphl 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 higl 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 oncentrations 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 ar 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 Ragl 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 Cell. Author manuscript; available in PMC 2016 April 09
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. Page 9 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript