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 09physiology 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