Microbiota-related Changes in Bile Acid & Tryptophan Metabolism are Associated with Gastrointestinal Dysfunction in a Mouse Model of Autism

The correlation between 5-HT tissue levels/intestinal transit rate and Firmicutes is mainly driven by the Blautia genus of the Clostridiales order.

“this correlation was mainly driven by the Blautia genus of the Clostridiales order (Fig. 2g).”

Minor Bacteroidetes genera such as Odoribacter, Parabacteroides, RC9 gut group and Rikenella show a reduction in relative abundance in BTBR mice.

“this seems to have happened at the expense of minor Bacteroidetes genera such as Odoribacter, Parabacteroides, RC9 gut group and Rikenella, which all showed a reduction in relative abundance.”

BTBR mice display decreased fecal water contents.

“BTBR mice displayed an elongated colon and decreased fecal water contents (Fig. 2b–c)”

Observed changes in GI physiology in BTBR mice are associated with a reduction in the abundance of certain bacterial taxa in the BTBR microbiota.

“We further associated the observed changes in GI physiology with a reduction in the abundance of certain bacterial taxa in the BTBR microbiota.”

Whole intestinal transit is significantly delayed in BTBR mice.

“we examined intestinal motility, and found that whole intestinal transit was significantly delayed in BTBR mice (Fig. 2a).”

The bloom in Bacteroidetes in BTBR mice is mainly driven by an increase in S24-7 uncultured bacterium.

“The bloom in Bacteroidetes was mainly driven by an increase in S24–7 uncultured bacterium, a dominant genus of S24–7 family.”

Expression levels of both Ido1 and Tdo2 genes were markedly down-regulated in the BTBR colon.

“We found that the expression levels of both Ido1 and Tdo2 genes were markedly down-regulated in the BTBR colon.”

It took almost twice as long to initiate spontaneous propagation of the pellet along the colon in BTBR mice.

“it took almost twice as long to initiate spontaneous propagation of the pellet along the colon”

Ex vivo analysis of colonic motility provides clear evidence of impaired transit in the BTBR large intestine.

“The follow-up ex vivo analysis of colonic motility provided clear evidence of impaired transit in the BTBR large intestine.”

BTBR mice have down-regulation of Tph1 gene expression in the intestine.

“which was coupled with a down-regulation of Tph1 and an up-regulation of Sert gene expression (Fig. 2e–f).”

The ratio between 5-HIAA and 5-HT tended to be increased in the BTBR intestine.

“the ratio between 5-HIAA (the major metabolite of 5-HT) and 5-HT tended to be increased in the BTBR intestine (Fig. 2e–f).”

Opposite changes in Ido1 and Tdo2 genes were observed in the small intestine of BTBR mice.

“Interestingly, opposite changes in Ido1 and Tod2 genes were observed in the small intestine of BTBR mice (Fig. 2e–f).”

BTBR mice have up-regulation of Sert gene expression in the intestine.

“which was coupled with a down-regulation of Tph1 and an up-regulation of Sert gene expression (Fig. 2e–f).”

The decrease in Firmicutes in BTBR mice is largely driven by the Lachnospiraceae family, in particular Blautia bacteria, but also Lachnospiraceae_Incertae Sedis and Coprococcus.

“A decrease in Firmicutes was largely driven by Lachnospiraceae family, in particular Blautia bacteria, but also Lachnospiraceae_Incertae Sedis and Coprococcus.”

BTBR mice display significant changes in short chain fatty acid levels in the gut lumen.

“BTBR mice also displayed significant changes in the SCFAs levels in the gut lumen (Fig. 1d).”

BTBR mice have a 50% reduction in 5-HT tissue levels in both small and large intestine.

“We observed a 50% reduction in 5-HT tissue levels in both small and large intestine of BTBR mice”

BTBR cecum has a substantial reduction in bacterial species diversity.

“The 16S sequencing revealed a substantial reduction in the bacterial species diversity in the BTBR cecum (Fig. 1a).”

There was a trend towards a reduction in the velocity of oral-to-anal propulsion in BTBR mice.

“there was a trend towards a reduction in the velocity of oral-to-anal propulsion in BTBR mice (Fig. 2d).”

BTBR and C57BL/6 microbiotas are clearly separated by principal coordinate analysis.

“Principal coordinate analysis showed a clear separation of BTBR and C57BL/6 microbiotas (Fig. 1b).”

BTBR mice display a distinct shift towards an increase in Bacteroidetes and a decrease in Firmicutes at the phylum level.

“At the phylum level, BTBR mice displayed a distinct shift towards an increase in Bacteroidetes and a decrease in Firmicutes; these are two major bacterial phyla altogether comprising > 90% of murine intestinal microbiota (Fig. 1c).”

BTBR mice display an elongated colon.

“BTBR mice displayed an elongated colon and decreased fecal water contents (Fig. 2b–c)”

There are strong correlations between the abundance of Blautia and Tph1, Sert and Ido1 gene expression levels.

“There were also strong correlations between the abundance of Blautia and Tph1, Sert and Ido1 gene expression levels (Table S3).”

Downstream targets of FXR, in particular Fgf15 and Nos2, were substantially down-regulated in BTBR ileum.

“However, the downstream targets of this receptor, in particular Fgf15 and Nos2, were substantially down-regulated.”

In the distal ileum, BTBR mice show a dramatic increase in epithelial permeability.

“In the distal ileum, we observed a dramatic increase in epithelial permeability in BTBR mice (Fig. 4a).”

BTBR mice display enhanced engagement in repetitive behaviors.

“In agreement with previous reports (Meyza et al., 2013), BTBR mice demonstrated impaired social interactions (a–c) and enhanced engagement in repetitive behaviors (e–f), as well as increased anxiety levels (h–k).”

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There is a strong positive correlation between FITC flux and S24-7 uncultured bacterium abundance in BTBR mice.

“In Bacteroidetes, a strong positive correlation between FITC flux and S24–7 uncultured bacterium abundance was present.”

BTBR ileum shows down-regulation of Slc5a1 gene.

“we observed a down-regulation of Slc5a1 gene in the BTBR ileum (Fig. S2a).”

Expression levels of both Pgc-1α and Pparγ genes were decreased in the BTBR liver.

“Similar to Cyp7a1, the expression levels of both Pgc-1α and Pparγ genes were decreased in the BTBR liver (Fig. 5c)”

Similar, but weaker associations were observed with the Lachnospiraceae Incertae Sedis genus.

“Similar, but weaker associations were observed with the Lachnospiraceae Incertae Sedis genus of the same order.”

BTBR mice have a 50% reduction in the numbers of inhibitory motor neurons (nNOS).

“we found a 50% reduction in the numbers of inhibitory motor neurons (nNOS) and total myenteric neurons (pan-neuronal HuC/HuD marker) (Fig. 3c–d).”

In both small and large intestine, Isc was substantially decreased in BTBR mice.

“In both small and large intestine, Isc was substantially decreased in BTBR mice (Fig. 4f, right panel).”

Morphometric analysis revealed a substantial reduction in the spatial density of MP in BTBR mice: the area occupied by MP was decreased.

“The morphometric analysis revealed a substantial reduction in the spatial density of MP in BTBR mice: the area occupied by MP was decreased, while the interganglionic space was increased (Fig. 3b).”

The ratio between secondary and primary unconjugated BAs was significantly decreased in BTBR mice.

“the ratio between secondary and primary unconjugated BAs was significantly decreased in BTBR mice (Fig. S3).”

BTBR mice show no substantial alterations in gene expression levels of TJ proteins (Cldn1, Ocln and Tjp1) or proteins regulating assembly and energy supply to TJs (Ckb, Ckm, Mylk) in distal ileum.

“Despite an increase in FITC flux, we failed to find substantial alterations in the gene expression levels of either TJ proteins (Cldn1, Ocln and Tjp1) or proteins regulating the assembly and energy supply to TJs (Ckb, Ckm, Mylk) (Glover et al., 2013; Shen et al., 2006) (Fig. 4b).”

In plasma, the levels of tauro-conjugated and unconjugated BAs were overall decreased in BTBR mice.

“In plasma, the levels of tauro-conjugated and unconjugated BAs were overall decreased in BTBR mice (Fig. 5a).”

In the fecal matter of BTBR mice, there is a remarkable increase in all conjugated BAs, for both primary and secondary conjugates.

“In the fecal matter of BTBR mice, we observed a remarkable increase in all conjugated BAs, for both primary and secondary conjugates (Fig. 5d, right panel).”

During the first 90 min of the assay, FITC flux was slightly decreased in BTBR colon.

“During the first 90 min of the assay, FITC flux was slightly decreased (p = 0.002 for the time × genotype interaction, F(6;102) = 9.198; p = 0.003 for 60 min and p = 0.057 for 90 min).”

BTBR mice have a 50% reduction in total myenteric neurons (HuC/HuD).

“we found a 50% reduction in the numbers of inhibitory motor neurons (nNOS) and total myenteric neurons (pan-neuronal HuC/HuD marker) (Fig. 3c–d).”

In BTBR mice, correlations with Blautia genus were largely lost for intestinal permeability.

“From the Firmicutes phylum, correlations with Blautia genus were largely lost.”

Expression levels of Fxr gene, as well as its partners Rxrα and Hnf4α, were unchanged in the BTBR ileum.

“The expression levels of Fxr gene, as well as its partners Rxrα and Hnf4α, were unchanged in the BTBR ileum (Fig. 5b).”

Colonic tissue in BTBR mice shows a very distinct profile of macromolecular permeability.

“Colonic tissue showed a very distinct profile of macromolecular permeability in BTBR mice (Fig. 4c).”

BTBR mice display impaired social interactions.

“In agreement with previous reports (Meyza et al., 2013), BTBR mice demonstrated impaired social interactions (a–c) and enhanced engagement in repetitive behaviors (e–f), as well as increased anxiety levels (h–k).”

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Expression of Cyp7b1 gene was up-regulated in BTBR mice.

“the expression of Cyp7b1 gene, which encodes a rate-limiting enzyme in the alternative pathway of BA synthesis, was up-regulated in BTBR mice.”

There is a strong negative association between the abundance of Ruminococcaceae Incertae Sedis bacteria and FITC permeability in BTBR mice.

“Instead, a strong negative association between the abundance of Ruminococcaceae Incertae Sedis bacteria and FITC permeability was observed.”

There was a trend towards a reduction in the expression of Cyp7a1 gene in BTBR liver.

“there was a trend towards a reduction in the expression of Cyp7a1 gene, which encodes a rate-limiting enzyme in the classical pathway of BA synthesis in the liver (Fig. 5c).”

The interganglionic space was increased in BTBR mice.

“The morphometric analysis revealed a substantial reduction in the spatial density of MP in BTBR mice: the area occupied by MP was decreased, while the interganglionic space was increased (Fig. 3b).”

BTBR colon has a 50% down-regulation of creatine kinase Ckb and Ckm genes.

“a 50% down-regulation of creatine kinase Ckb and Ckm genes suggests a reduced efficacy of phosphocreatine metabolic shuttle turnover”

There are no changes in the gene expression of intestinal BA transporters (Asbt, Fabp6, Ostα) or liver BA transporters (Ntcp, Bsep) across the groups.

“We did not find any changes in the gene expression of intestinal BA transporters (Asbt, Fabp6, Ostα) or liver BA transporters (Ntcp, Bsep) across the groups (Fig. 5b–c).”

Only marginal changes in ion conductance (TEER) were observed in either ileum or colon samples of BTBR mice.

“Despite marked alterations in FITC permeability, only marginal changes in ion conductance (TEER) were observed in either ileum or colon samples of BTBR mice (Fig. 4f, left panel).”

The expression levels of Ido1 and Tdo2 genes are markedly altered in the BTBR intestine.

“The expression levels of Ido1 and Tdo2 genes, which encode alternative rate-limiting enzymes in this pathway, were markedly altered in the BTBR intestine (Fig. 2e–f).”

Behavioral scores are associated with changes in intestinal serotonin levels and other GI parameters assessed.

“Given the tight interactions between the gut microbiota and GI functions (Table S3), it is perhaps not surprising that behavioral scores were also associated with changes in the intestinal serotonin levels and other GI parameters assessed (Fig. S4).”

BTBR mice display increased anxiety levels.

“In agreement with previous reports (Meyza et al., 2013), BTBR mice demonstrated impaired social interactions (a–c) and enhanced engagement in repetitive behaviors (e–f), as well as increased anxiety levels (h–k).”

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Intestinal 5-HT levels and Tph1 gene expression are strongly and positively correlated with the relative abundance of Blautia genus.

“Strikingly, intestinal 5-HT levels and Tph1 gene expression were strongly and positively correlated with the relative abundance of Blautia genus (Fig. 2g, Table S3).”

There is a reduction in the secondary BA pool in BTBR fecal matter.

“First, we observed a dramatic increase in all conjugated BAs, as well as a reduction in secondary BA pool in BTBR fecal matter (Fig. 5d and Fig. S3).”

BTBR mice display a reduction in plasma BA levels.

“In addition to the deficits in bacteria-mediated bile conversion, BTBR mice displayed a reduction in plasma BA levels, most likely due to a decrease in BA synthesis along the classical Cyp7a1-mediated pathway (Fig. 5a and c).”

BTBR mice have elevated plasma corticosterone at the peak of the acute stress response.

“Further, BTBR mice displayed higher baseline levels of the stress hormone corticosterone, as well as elevated corticosterone values at the peak of the acute stress response and during the subsequent recovery phase (Fig. 6l, p < 0.05 for the effect of genotype, F(1;16) = 8.193).”

Bilophila and Bacteroides species are increased in abundance in BTBR mice.

“For the S24–7 family, Bilophila and Bacteroides species (all increased in abundance in BTBR mice), a negative association with sociability levels and a positive association with compulsive and anxiety-like behaviors was observed (Fig. 7).”

BTBR mice have smaller adrenal glands.

“Despite this hyper-corticosterone phenotype, BTBR mice had smaller adrenal glands, and increased expression of the glucocorticoid receptor gene in the hippocampus (Fig. 6m–n).”

There is a dramatic increase in all conjugated BAs in BTBR fecal matter.

“First, we observed a dramatic increase in all conjugated BAs, as well as a reduction in secondary BA pool in BTBR fecal matter (Fig. 5d and Fig. S3).”

Rikenella, Parabacteroides, Odoribacter, Desulfovibrio, Blautia and Bifidobacterium species are reduced in abundance in BTBR mice.

“Rikenella, Parabacteroides, Odoribacter, Desulfovibrio, Blautia and Bifidobacterium species (all reduced in abundance in BTBR mice) were positively correlated with the social behavior scores and negatively correlated with the repetitive behaviors and anxiety phenotype.”

BTBR mice have elevated plasma corticosterone during the subsequent recovery phase.

“Further, BTBR mice displayed higher baseline levels of the stress hormone corticosterone, as well as elevated corticosterone values at the peak of the acute stress response and during the subsequent recovery phase (Fig. 6l, p < 0.05 for the effect of genotype, F(1;16) = 8.193).”

Lactobacilli tend to be decreased in the BTBR gut.

“Of note, lactobacilli also tended to be decreased in the BTBR gut (Table S2).”

BTBR mice have decreased plasma levels of oxytocin.

“Here we found decreased levels of social behavior-related hormone oxytocin in BTBR plasma (d).”

• Also stated in: § Discussion · plain

  “Interestingly, we found that deficits in social behavior in BTBR mice were accompanied by a decrease in plasma oxytocin levels (Fig. 6d).”

There is a strong association between the gut microbiota and the behavioral phenotype.

“Correlation analysis between the relative abundance of bacterial taxa and behavioral scores showed an impressively strong association between the gut microbiota and the behavioral phenotype (Fig. 7).”

BTBR mice have increased expression of the glucocorticoid receptor gene in the hippocampus.

“Despite this hyper-corticosterone phenotype, BTBR mice had smaller adrenal glands, and increased expression of the glucocorticoid receptor gene in the hippocampus (Fig. 6m–n).”

Cyp11a1 gene expression is increased in the BTBR ileum.

“Strikingly, we found an increased expression of Cyp11a1 gene in the BTBR ileum (Fig. S2b), which could have resulted in elevated production of corticosterone and an up-regulation of Tdo2 locally in the BTBR ileum (Fig. 2f).”

There is a marked reduction in 5-HT bioavailability in BTBR small and large intestine.

“Secondly, we found a marked reduction in 5-HT bioavailability in BTBR small and large intestine due to a decrease in 5-HT synthesis and an increase in 5-HT breakdown (Fig. 2e–f).”

Cyp11a1 expression is substantially up-regulated in the distal ileum of BTBR mice.

“Interestingly, the expression of Cyp11a1, a gene encoding the first rate-limiting enzyme in the production of corticosterone, was substantially up-regulated in the distal ileum of BTBR mice (Fig. S2b).”

BTBR mice display profound changes in the cecal microbiota, with 18 out of 44 identified bacterial genera being differentially altered.

“BTBR mice displayed profound changes in the cecal microbiota, with 18 out of 44 identified bacterial genera being differentially altered (Table S2).”

iNos (Nos2) gene expression is down-regulated in the ileum of BTBR mice.

“A down-regulation of iNos (Nos2) gene expression in the ileum of BTBR mice (Fig. 5b) suggests that anti-microbial defense can be compromised in the BTBR intestine, which in turn could contribute to the impairment of small intestine barrier function (Fig. 4a).”

BTBR mice have higher baseline plasma corticosterone levels.

“Further, BTBR mice displayed higher baseline levels of the stress hormone corticosterone, as well as elevated corticosterone values at the peak of the acute stress response and during the subsequent recovery phase (Fig. 6l, p < 0.05 for the effect of genotype, F(1;16) = 8.193).”

The BTBR myenteric plexus shows reduced numbers of excitatory and inhibitory neurons.

“Further, the BTBR myeneric plexus shows reduced numbers of excitatory and inhibitory neurons.”

Gut microbes, in particular spore-forming clostridia, have been shown to regulate 5-HT metabolism in the GI tract.

“Gut microbes, in particular spore-forming clostridia, have been shown to regulate 5-HT metabolism in the GI tract (Yano et al., 2015).”

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The propagation of fecal matter along the colon is substantially slowed in BTBR mice.

“both observations suggesting that the propagation of fecal matter along the colon was substantially slowed.”

Release of mucosal serotonin (5-HT) triggers intestinal peristalsis.

“The release of mucosal serotonin (5-HT) triggers intestinal peristalsis (Heredia et al., 2013).”

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BTBR mice have a very robust phenotype of the gut microbiota.

“Overall, BTBR mice seem to have a very robust phenotype of the gut microbiota.”

Changes in bile acid and tryptophan metabolism are putative mechanisms underlying GI dysfunction in BTBR mice.

“We identified changes in bile acid and tryptophan metabolism as putative mechanisms underlying GI dysfunction in these animals.”

ASD subjects display substantial alterations in microbiota composition.

“There is a growing body of evidence that ASD subjects display substantial alterations in microbiota composition, along with marked symptoms of GI distress, including changes to bowel habits, increased intestinal permeability, bloating and abdominal pain (Angelis et al., 2013; D'Eufemia et al., 1996; Kang et al., 2013; Luna et al., 2017; McElhanon et al., 2014; Tomova et al., 2015).”

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These findings rule out BA malabsorption in BTBR mice.

“These findings rule out the BA malabsorption in BTBR mice.”

This would favor a rapid increase in macromolecular permeability in BTBR samples in ex vivo conditions, when tissue specimens are exposed to limited oxygen and substrate supply.

“This would favor a rapid increase in macromolecular permeability in BTBR samples in ex vivo conditions, when tissue specimens are exposed to limited oxygen and substrate supply.”

Gut bacteria and their metabolites can modulate intestinal barrier function.

“Gut bacteria and their metabolites can modulate intestinal barrier function (Kelly et al., 2015);”

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Epithelial permeability data clearly demonstrated functional differences between the colon and the ileum in BTBR mice.

“Epithelial permeability data (Fig. 4) clearly demonstrated functional differences between the colon and the ileum in BTBR mice.”

These data suggest an abnormal development of both excitatory and inhibitory motor neurons in BTBR mice.

“These data suggest an abnormal development of both excitatory and inhibitory motor neurons in BTBR mice.”

These findings suggest that the BTBR colon might be less resilient to any metabolic stress in vivo, such as intestinal inflammation or hypoxia.

“Furthermore, these findings suggest that the BTBR colon might be less resilient to any metabolic stress in vivo, such as intestinal inflammation or hypoxia.”

A decrease in plasma BAs in BTBR mice could be attributed to a deficit in either BA synthesis or BA re-uptake.

“Thus, a decrease in plasma BAs in BTBR mice could be attributed to a deficit in either BA synthesis or BA re-uptake.”

• AssumptionRests on an assumption: About 95% of the bile released into the gut is actively re-absorbed in distal ileum to ensure the complete enterohepatic circulation.

A reduction in Cl− secretion in the BTBR colon would be consistent with decreased fecal water contents and delayed colonic transit.

“In this regard, a reduction in Cl− secretion in the BTBR colon would be consistent with decreased fecal water contents and delayed colonic transit (Fig. 2b–d).”

It is surprising that the suppression of the intestinal Fxr/Fgf15 signaling pathway failed to up-regulate Cyp7a1 gene expression in the liver of BTBR mice.

“Hence, it is surprising that the suppression of the intestinal Fxr/Fgf15 signaling pathway failed to up-regulate Cyp7a1 gene expression in the liver of BTBR mice.”

• AssumptionRests on an assumption: When absorbed in distal ileum, BAs activate the farnesoid X receptor (FXR) in enterocytes.

These data indicate that the BTBR microbiota has a reduced capacity to metabolize host bile.

“These data indicate that the BTBR microbiota has a reduced capacity to metabolize host bile.”

• AssumptionRests on an assumption: Gut bacteria de-conjugate BAs from taurine and glycine.
• AssumptionRests on an assumption: Unconjugated primary BAs (CA, CDCA and MCA in mice) are processed into secondary BAs, such as DCA, LCA and UDCA.

A marked increase in Cldn1 gene expression can explain a functionally 'tighter' epithelium in the BTBR colon at baseline.

“On one hand, a marked increase in Cldn1 gene expression can explain a functionally “tighter” epithelium in the BTBR colon at baseline.”

Multiple nuclear receptors are implicated in the decreased BA synthesis observed in BTBR mice.

“suggesting that multiple nuclear receptors are implicated in the decreased BA synthesis observed in BTBR mice.”

Intestinal Fgf15 is a potent inhibitor of Cyp7a1 expression and BA synthesis.

“Intestinal Fgf15 is a potent inhibitor of Cyp7a1 expression and BA synthesis (Kong et al., 2012).”

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The 50% down-regulation of Ckb and Ckm genes suggests a reduced efficacy of phosphocreatine metabolic shuttle turnover, which is critically important for the maintenance of energy supply to TJs.

“a 50% down-regulation of creatine kinase Ckb and Ckm genes suggests a reduced efficacy of phosphocreatine metabolic shuttle turnover, which is critically important for the maintenance of energy supply to TJs (Glover et al., 2013).”

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Secondary BAs activate colonic motility by stimulating the release of intestinal 5-HT and CGRP.

“For example, secondary BAs were shown to activate colonic motility by stimulating the release of intestinal 5-HT and CGRP (Alemi et al., 2013).”

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Unconjugated primary BAs are transformed into secondary BAs by 7α-dehydroxylation activity present in some members of Clostridium cluster XIVa including Blautia species.

“Unconjugated primary BAs are further transformed into secondary BAs as a result of 7α-dehydroxylation activity, which is present in some members of Clostridium cluster XIVa including Blautia species (Ridlon et al., 2013).”

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An increase in Tβ-MCA intestinal levels can explain the observed down-regulation of Fgf15 gene expression in the BTBR ileum.

“Hence, an increase in Tβ-MCA intestinal levels can explain the observed down-regulation of Fgf15 gene expression in the BTBR ileum (Fig. 5b and d).”

Metabolism of tryptophan along the kynurenine route seems to be affected in BTBR mice.

“Interestingly, metabolism of tryptophan along the kynurenine route also seems to be affected in BTBR mice.”

Secondary BAs have a unique signaling profile in the GI tract compared with primary bile moieties.

“Secondary BAs, produced by gut bacteria, have a unique signaling profile in the GI tract as compared with primary bile moieties.”

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The observed reduction in the abundance of Blautia species could underlie the deficit in 5-HT production in the BTBR gut.

“Hence, the observed reduction in the abundance of Blautia species could underlie the deficit in 5-HT production in the BTBR gut.”

Anti-microbial defense can be compromised in the BTBR intestine.

“A down-regulation of iNos (Nos2) gene expression in the ileum of BTBR mice (Fig. 5b) suggests that anti-microbial defense can be compromised in the BTBR intestine, which in turn could contribute to the impairment of small intestine barrier function (Fig. 4a).”

Dysfunctional intestinal barrier in BTBR mice can permit the microbiota to induce low-grade inflammation in the gut.

“Dysfunctional intestinal barrier in BTBR mice can permit the microbiota to induce low-grade inflammation in the gut.”

Compromised anti-microbial defense could contribute to the impairment of small intestine barrier function in BTBR mice.

“A down-regulation of iNos (Nos2) gene expression in the ileum of BTBR mice (Fig. 5b) suggests that anti-microbial defense can be compromised in the BTBR intestine, which in turn could contribute to the impairment of small intestine barrier function (Fig. 4a).”

The reduction in plasma BA levels in BTBR mice is most likely due to a decrease in BA synthesis along the classical Cyp7a1-mediated pathway.

“In addition to the deficits in bacteria-mediated bile conversion, BTBR mice displayed a reduction in plasma BA levels, most likely due to a decrease in BA synthesis along the classical Cyp7a1-mediated pathway (Fig. 5a and c).”

A reduction in secondary BA levels could contribute to the delayed colonic transit in BTBR mice.

“Therefore, a reduction in secondary BA levels could contribute to the delayed colonic transit in BTBR mice (Fig. 2b–d).”

A decrease in 5-HT bioavailability can explain delayed intestinal transit and reduced activity of electrolyte transport seen in BTBR mice.

“Hence, a decrease in 5-HT bioavailability can explain delayed intestinal transit and reduced activity of electrolyte transport seen in BTBR mice (Figs. 2a–d, 4f).”

The observed changes in intestinal Ido1 and Tdo2 gene expression are very unlikely to have a direct effect on the brain in BTBR mice.

“The observed changes in intestinal Ido1 and Tdo2 gene expression are very unlikely to have a direct effect on the brain in BTBR mice.”

A shift towards a pro-inflammatory state, particularly with an anxious phenotype, can trigger activation of neuroinflammatory responses in the brain and exaggerate autistic-like behavioral deficits in BTBR mice.

“This shift towards a pro-inflammatory state, particularly in combination with an anxious phenotype (Fig. 6h–l), can in turn trigger the activation of neuroinflammatory responses in the brain (Rea et al., 2016; Wohleb et al., 2015) and exaggerate autistic-like behavioral deficits in BTBR mice.”

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The BTBR mouse has been shown to display a pro-inflammatory phenotype that is thought to contribute to the development of ASD-like behavior.

“Indeed, the BTBR mouse has been shown to display a pro-inflammatory phenotype that is thought to contribute to the development of ASD-like behavior (Careaga et al., 2015; Heo et al., 2011; Onore et al., 2013).”

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All these factors can cause delayed intestinal motility in BTBR mice.

“All this can cause delayed intestinal motility in BTBR mice.”

Deficient bacterial transformation of bile moieties and impaired tryptophan metabolism in the BTBR intestine are potential mechanisms underlying GI symptoms in these animals.

“We further identify deficient bacterial transformation of bile moieties and impaired tryptophan metabolism in the BTBR intestine as potential mechanisms underlying GI symptoms in these animals.”

Deficient synthesis of BAs exaggerates the effect of loss of Fxr signaling on bacterial overgrowth.

“This effect is further exaggerated by deficient synthesis of BAs, which normally prevent bacterial overgrowth.”

The BTBR mouse displays marked GI dysfunction.

“In summary, here we show that the BTBR mouse displays marked GI dysfunction (Fig. 8).”

Reduced abundance of bile-metabolizing Bifidobacterium species increases the fraction of tauro-conjugated BAs (e.g. TβMCA) in the GI tract.

“Reduced abundance of bile-metabolizing Bifidobacterium species increases the fraction of tauro-conjugated BAs (e.g. tauromuricholic acid (TβMCA)) in the GI tract.”

These functional changes are associated with a reduction in the relative abundance of Bifidobacterium and Blautia bacteria in the BTBR microbiota.

“Taking advantage of microbiota sequencing and physiological data, we associate these functional changes with a reduction in the relative abundance of Bifidobacterium and Blautia bacteria in the BTBR microbiota.”