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 09regression 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 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 Yano et al. Page 8 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript