Yano et al GF mice increases 5-HT staining colocalized to CgA+ ECs( Figure 2), elevates host coloni TPHI expression( Figure 3D)and decreases SLC6A4 expression(Figure 3E)toward levels seen in SPF mice. Improvements in serum 5-HT are observed within 2 days after inoculation of GF mice with Sp( Figure S2E), and do not correlate with amelioration of abnormal cecal weight( Figure S2F). Importantly, Sp also elevates colonic 5-HT in Ragl knockout mice (Figure S2G), which lack adaptive immune cells, indicating that the effects of Sp on gut 5- HT are not dependent on Sp-mediated regulatory T cell induction(Stefka et al., 2014) Notably, the 5-HT-promoting effects of Sp are recapitulated by colonization of GF mice ith spore-forming microbes from the healthy human colonic microbiota(hSp)(Figure S3) suggesting that the serotonergic function of this community is conserved across mice and humans To determine whether the effects of Sp on host 5-hT depend on colonic Tph activity, we colonized GF mice with Sp on P42 and then administered the Tph inhibitor para- c9 chlorophenylalanine(PCPA)intrarectally twice daily for 3 days prior to 5-HT assessments on P56 ( Liu et al., 2008). Intrarectal injection of PCPA sufficiently blocks the ability of Sp to elevate colon and serum 5-HT levels(Figures 3C and S2C), as well as Sp-mediated increases in 5-HT staining in ECs(Figure 2). Similar effects of PCPA treatment on blocking increases in colon 5-HT, serum 5-HT and 5-HT staining in colonic ECs are seen in GF mice colonized with hSp (Figure S3). Interestingly, inhibiting Tph activity with PCPA results in a compensatory increase in colonic TPHI and decrease in SLC6A4(Figures 3D and S2D) expression in Sp-colonized mice, supporting the notion that microbiota-dependent changes in 5-HT transporter levels occur as a secondary response to Tph modulation To further evaluate whether changes in SLC6A4 expression are necessary for microbiota- mediated alterations in peripheral 5-HT, we tested the effects of microbiota manipulations on colon and serum 5-HT in SLC6A4 heterozygous (+/-)and complete(-/-)knockout(KO) mice. Depleting the microbiota via P42-P56 antibiotic treatment(Reikvam et al., 2011)of SPF SLC6A4 +/-and-/-mice effectively decreases colonic 5-HT levels(Figures S4A and S4B), indicating that the microbiota is required for promoting gut 5-HT in SIc6a4-deficient mice. Colonizing antibiotic-treated SLC6A4 +/-and-/-mice with Sp raises colon 5-HT to levels seen in SPF SLC6A4+/-and-/-mice(Figure S4A), demonstrating that Slc6a4 is not required for conferring the effects of Sp on gut 5-HT. Antibiotic-induced decreases and Sp induced increases in colon 5-HT levels can be attributed to modulation of 5-ht content colonic ECs from SLC6A4 +/-and-/-mice(Figure $4C). Similar effects of antibiotic treatment and Sp colonization are seen for serum 5-HT in SLC6A4+/-mice, whereas SLC6A4-1-mice exhibit low to undetectable levels of serum 5-HT, highlighting the dependence of platelets on Slc6a4-mediated 5-HT uptake( Figure S4B). Taken together, these data support a role for Sp in promoting Tphl-mediated 5-HT biosynthesis by colonic ECS, regulating both colon and serum levels of 5-HT Microbiota-Mediated Regulation of Host Serotonin Modulates Gastrointestinal Motility Intestinal 5-HT plays an important role in stimulating the enteric nervous system and gl function( Gershon and Tack, 2007). To determine whether microbiota-dependent modulation of colonic 5-HT impacts GI motility, we colonized P42 GF mice with Sp and Cell. Author manuscript; available in PMC 2016 April 09GF mice increases 5-HT staining colocalized to CgA+ ECs (Figure 2), elevates host colonic TPH1 expression (Figure 3D) and decreases SLC6A4 expression (Figure 3E) toward levels seen in SPF mice. Improvements in serum 5-HT are observed within 2 days after inoculation of GF mice with Sp (Figure S2E), and do not correlate with amelioration of abnormal cecal weight (Figure S2F). Importantly, Sp also elevates colonic 5-HT in Rag1 knockout mice (Figure S2G), which lack adaptive immune cells, indicating that the effects of Sp on gut 5- HT are not dependent on Sp-mediated regulatory T cell induction (Stefka et al., 2014). Notably, the 5-HT-promoting effects of Sp are recapitulated by colonization of GF mice with spore-forming microbes from the healthy human colonic microbiota (hSp) (Figure S3), suggesting that the serotonergic function of this community is conserved across mice and humans. To determine whether the effects of Sp on host 5-HT depend on colonic Tph activity, we colonized GF mice with Sp on P42 and then administered the Tph inhibitor para￾chlorophenylalanine (PCPA) intrarectally twice daily for 3 days prior to 5-HT assessments on P56 (Liu et al., 2008). Intrarectal injection of PCPA sufficiently blocks the ability of Sp to elevate colon and serum 5-HT levels (Figures 3C and S2C), as well as Sp-mediated increases in 5-HT staining in ECs (Figure 2). Similar effects of PCPA treatment on blocking increases in colon 5-HT, serum 5-HT and 5-HT staining in colonic ECs are seen in GF mice colonized with hSp (Figure S3). Interestingly, inhibiting Tph activity with PCPA results in a compensatory increase in colonic TPH1 and decrease in SLC6A4 (Figures 3D and S2D) expression in Sp-colonized mice, supporting the notion that microbiota-dependent changes in 5-HT transporter levels occur as a secondary response to Tph modulation. To further evaluate whether changes in SLC6A4 expression are necessary for microbiota￾mediated alterations in peripheral 5-HT, we tested the effects of microbiota manipulations on colon and serum 5-HT in SLC6A4 heterozygous (+/−) and complete (−/−) knockout (KO) mice. Depleting the microbiota via P42-P56 antibiotic treatment (Reikvam et al., 2011) of SPF SLC6A4 +/− and −/− mice effectively decreases colonic 5-HT levels (Figures S4A and S4B), indicating that the microbiota is required for promoting gut 5-HT in Slc6a4-deficient mice. Colonizing antibiotic-treated SLC6A4 +/− and −/− mice with Sp raises colon 5-HT to levels seen in SPF SLC6A4 +/− and −/− mice (Figure S4A), demonstrating that Slc6a4 is not required for conferring the effects of Sp on gut 5-HT. Antibiotic-induced decreases and Sp￾induced increases in colon 5-HT levels can be attributed to modulation of 5-HT content in colonic ECs from SLC6A4 +/− and −/− mice (Figure S4C). Similar effects of antibiotic treatment and Sp colonization are seen for serum 5-HT in SLC6A4 +/− mice, whereas SLC6A4 −/− mice exhibit low to undetectable levels of serum 5-HT, highlighting the dependence of platelets on Slc6a4-mediated 5-HT uptake (Figure S4B). Taken together, these data support a role for Sp in promoting Tph1-mediated 5-HT biosynthesis by colonic ECs, regulating both colon and serum levels of 5-HT. Microbiota-Mediated Regulation of Host Serotonin Modulates Gastrointestinal Motility Intestinal 5-HT plays an important role in stimulating the enteric nervous system and GI function (Gershon and Tack, 2007). To determine whether microbiota-dependent modulation of colonic 5-HT impacts GI motility, we colonized P42 GF mice with Sp and Yano et al. Page 5 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript