Yano et al platelets isolated from GF mice exhibit impaired aggregation in response to in vitro collagen stimulation, as measured by decreased levels of high granularity, high mass aggregates detected by both flow cytometry(De Cuyper et al., 2013, Nieswandt et al., 2004)(Figures 5B 5C, S5C an S5D)and imaging( Figure S5B). Colonization of GF mice with Sp restores levels of platelet aggregation to those seen in SPF mice. These effects of Sp on correcting impaired platelet aggregation are attenuated by colonic PCPA injection, indicating dependence on Tph activity. Overall, these findings suggest that Sp-mediated elevations in colonic 5-HT, and thus platelet 5-HT, promote platelet activation and aggregation relevant to hemostasis Microbial metabolites mediate Effects of the microbiota on host serotonin In light of the important role for Sp in regulating 5-HT-related intestinal and platelet function, we aimed to identify specific microbial factors responsible for conferring the serotonergic effects of Sp. Based on our finding that Sp elevates 5-HT particularly in colonic c9 ECs(Figure 2), we hypothesized that Sp promotes levels of a soluble factor that signal directly to ECs to modulate TPHI expression and 5-HT biosynthesis. To test this, we prepared filtrates of total colonic luminal contents from Sp-colonized mice and controls, and evaluated their effects on levels of 5-HT in RiN14B chromaffin cell cultures(Nozawa et al 2009) Relative to vehicle-treated controls, there is no significant effect of filtered colonic uminal contents from GF mice on levels of 5-HT released or TPHl expressed from RIN14B cells( Figures 6A and 6B). Filtered colonic luminal contents from SPF and Sp-colonized mice sufficiently induce 5-HT from RIN14B cells (Figure 6A), to levels comparable to those elicited by the calcium ionophore, ionomycin, as a positive control. TPHI expression is also elevated in chromaffin cells exposed to SPF and Sp luminal filtrates, suggesting increased 5- HT synthesis. This is in contrast to ionomycin, which stimulates 5-HT release, but has no ffect on TPHl expression, from RIN14B cells Importantly, these findings suggest that microbiota-mediated increases in gut 5-HT are conferred via direct signaling of a soluble, dulated factor to colonic ecs We utilized metabolomic profiling to identify candidate Sp-dependent, 5-HT-inducing molecules in feces from adult mice. Sp colonization of GF mice leads to statistically ignificant alterations in 75% of the 416 metabolites detected, of which 76% are elevated and 24% are reduced, relative to vehicle-treated gF controls (Tables SI and S2). Similar changes are seen with hSp colonization, leading to co-clustering of Sp and hSp samples by principal components analysis(PCA)(Figure 6C). ASF colonization has a mild effect, significantly modulating 50%of metabolites detected(66% increased, 36%decreased (Table S2), and forming a distinct but proximal cluster to GF controls by PCA (Figure 6C) Postnatal conventionalization of GF mice with an SPf microbiota alters 66% of all metabolites detected (59% increased, 41% decreased)(Table S2), and produces substantial changes in the metabolome that are distinguishable from the effects of Sp, hSp and AsF along PC2 (Figure 6C). Notably, Sp, hSp and SPF colonization results in similar shifts along PCl, compared to vehicle and AsF-treated controls, suggesting common metabolic alterations among communities that similarly elevate peripheral 5-HT levels. Metabolomics profiling confirms that fecal 5-HT is commonly upregulated in the Sp, hSp and SPF fecal metabolome, and comparatively low in ASF and GF samples(Table S1). Simple linear Cell. Author manuscript; available in PMC 2016 April 09platelets isolated from GF mice exhibit impaired aggregation in response to in vitro collagen stimulation, as measured by decreased levels of high granularity, high mass aggregates detected by both flow cytometry (De Cuyper et al., 2013; Nieswandt et al., 2004) (Figures 5B 5C, S5C an S5D) and imaging (Figure S5B). Colonization of GF mice with Sp restores levels of platelet aggregation to those seen in SPF mice. These effects of Sp on correcting impaired platelet aggregation are attenuated by colonic PCPA injection, indicating dependence on Tph activity. Overall, these findings suggest that Sp-mediated elevations in colonic 5-HT, and thus platelet 5-HT, promote platelet activation and aggregation relevant to hemostasis. Microbial Metabolites Mediate Effects of the Microbiota on Host Serotonin In light of the important role for Sp in regulating 5-HT-related intestinal and platelet function, we aimed to identify specific microbial factors responsible for conferring the serotonergic effects of Sp. Based on our finding that Sp elevates 5-HT particularly in colonic ECs (Figure 2), we hypothesized that Sp promotes levels of a soluble factor that signals directly to ECs to modulate TPH1 expression and 5-HT biosynthesis. To test this, we prepared filtrates of total colonic luminal contents from Sp-colonized mice and controls, and evaluated their effects on levels of 5-HT in RIN14B chromaffin cell cultures (Nozawa et al., 2009). Relative to vehicle-treated controls, there is no significant effect of filtered colonic luminal contents from GF mice on levels of 5-HT released or TPH1 expressed from RIN14B cells (Figures 6A and 6B). Filtered colonic luminal contents from SPF and Sp-colonized mice sufficiently induce 5-HT from RIN14B cells (Figure 6A), to levels comparable to those elicited by the calcium ionophore, ionomycin, as a positive control. TPH1 expression is also elevated in chromaffin cells exposed to SPF and Sp luminal filtrates, suggesting increased 5- HT synthesis. This is in contrast to ionomycin, which stimulates 5-HT release, but has no effect on TPH1 expression, from RIN14B cells. Importantly, these findings suggest that microbiota-mediated increases in gut 5-HT are conferred via direct signaling of a soluble, Sp-modulated factor to colonic ECs. We utilized metabolomic profiling to identify candidate Sp-dependent, 5-HT-inducing molecules in feces from adult mice. Sp colonization of GF mice leads to statistically significant alterations in 75% of the 416 metabolites detected, of which 76% are elevated and 24% are reduced, relative to vehicle-treated GF controls (Tables S1 and S2). Similar changes are seen with hSp colonization, leading to co-clustering of Sp and hSp samples by principal components analysis (PCA) (Figure 6C). ASF colonization has a mild effect, significantly modulating 50% of metabolites detected (66% increased, 36% decreased) (Table S2), and forming a distinct but proximal cluster to GF controls by PCA (Figure 6C). Postnatal conventionalization of GF mice with an SPF microbiota alters 66% of all metabolites detected (59% increased, 41% decreased) (Table S2), and produces substantial changes in the metabolome that are distinguishable from the effects of Sp, hSp and ASF along PC2 (Figure 6C). Notably, Sp, hSp and SPF colonization results in similar shifts along PC1, compared to vehicle and ASF-treated controls, suggesting common metabolic alterations among communities that similarly elevate peripheral 5-HT levels. Metabolomics profiling confirms that fecal 5-HT is commonly upregulated in the Sp, hSp and SPF fecal metabolome, and comparatively low in ASF and GF samples (Table S1). Simple linear Yano et al. Page 7 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript