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 09and 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 Yano et al. Page 2 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript