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 09serve 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. 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