Microbially modified bile acids (DCA & CDCA) oscillate clock controlled genes in synchronised Caco-2 cells.

<p>Caco-2 cells were synchronized via serum starvation followed by a serum shock and treated with bile acids at 100 μM or with their corresponding bile salts. The cells were harvested for every 6h for a total of 48 hours. The relative expression levels of clock-regulated genes were measured using qRT-PCR and plotted in the graph versus time. The red colour graph represents the vehicle, pink represents the TDCA, brown represents TCDCA, blue represents DCA and green represents CDCA. Data represents pooled results from three independent biological replicates with two technical replicates.</p

Model by which microbially-modified bile acids may influence expression of circadian genes.

<p>Bile acids are synthesised from cholesterol and conjugated with taurine or glycine in the liver and stored in the gall bladder. Upon food intake, the bile salts are released into the duodenum and aid in fat digestion and adsorption. Gut microbes in the intestinal lumen deconjugate bile salts to yield unconjugated bile acids. Recent work from other groups has demonstrated that diet, antibiotics and probiotics may influence this microbial activity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167319#sec012" target="_blank">discussion</a>). Within the ileal enterocytes, unconjugated bile acids influence the amplitude and periodicity of circadian gene expression. Nearly 95% of bile salts and bile acids are reabsorbed in the terminal ileum and transported back to the liver via the hepatic portal circulation. Upon reaching the liver, the bile acids further influence circadian gene expression profiles.</p

Unconjugated bile acids (DCA & CDCA) influence expression of clock related genes in murine peripheral organs.

<p>C57BL/6 mice were given vehicle (Corn oil) or DCA or CDCA each at 9 μmol/kg bodyweight dissolved in corn oil via oral gavage. The mice were gavaged three times (t = 0, t = 24h and t = 48h) with corn oil or bile acids and were fasted for 3h prior to harvesting of tissues from (A, B) Ileum, (C, D) Colon and (E, F) Liver. All tissues were harvested within the same 45 minute window during the light phase to minimize variation between subjects (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167319#sec002" target="_blank">Materials & Methods</a>). Total RNA was isolated from tissues and mRNA expression was measured using qRT-PCR. The expression of clock-regulated genes was analysed. Data are plotted relative to β-actin expression. The white bar represents the results from vehicle, grey bar represents the DCA treatment group and the black bar represents the CDCA group. Error bars denote SEM. Statistical significance determined by one way ANOVA (*P<0.05, **P<0.01, ***P<0.001), n = 4.</p

Unconjugated bile acids (DCA and CDCA) influence expression of genes encoding input regulators of clock-regulated genes in murine peripheral organs.

<p>C57BL/6 mice were given vehicle (Corn oil) or DCA or CDCA each at 9 μmol/ kg bodyweight dissolved in corn oil via oral gavage. The mice were gavaged three times (t = 0, t = 24h and t = 48h) with corn oil or bile acids and were fasted for 3h prior to harvesting of tissues from (A, B) Ileum, (C, D) Colon and (E, F) Liver. All tissues were harvested within the same 45 minute window during the light phase to minimize variation between subjects (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167319#sec002" target="_blank">Materials & Methods</a>). Total RNA was isolated from tissues and mRNA expression was measured using qRT-PCR. The expression of clock-controlled genes was analysed. Data are plotted relative to β-actin expression. The white bar represents the results from vehicle, grey bar represents the DCA treatment group and the black bar represents the CDCA group. Error bars denote SEM. Statistical significance was determined by one way ANOVA (*P<0.05, **P<0.01, ***P<0.001), n = 4.</p

<i>B. infantis</i> attenuates NF-κB activation <i>in vivo</i> in response to <i>S. typhimurium</i> infection.

<p>(A) A representative in vivo image illustrates that NF-κB activation in NF-κBlux transgenic mice four hours following <i>S. typhimurium</i> infection is attenuated when an animal is pre-fed <i>B. infantis</i>; (B) <i>In vivo</i> NF-κB activation in <i>B. infantis</i>-fed animals was significant reduced compared to NF-κB activation in placebo-fed controls (n = 6/group). *p<0.05 versus placebo.</p

<i>B. infantis</i> reduces <i>S. typhimurium</i> disease severity and systemic translocation.

<p>(A) Macroscopic clinical scoring of mice infected with <i>S. typhimurium</i> reveals a significant reduction in <i>B. infantis</i> pre-fed mice disease symptoms 8 days following initial infection (n = 10/group); (B) <i>S. typhimurium</i> numbers were reduced in the spleens and livers of <i>B. infantis</i> fed mice 6 days following infection (n = 18/group). *p<0.05 versus placebo.</p

NF-κB activation & TNF-α secretion in response to LPS is significantly reduced by adoptive transfer of CD4⁺CD25⁺ T cells from <i>B. infantis</i>-fed animals.

<p>(A) In vivo adoptive transfer of CD4⁺ NF-κB^−/− T cells from <i>B. infantis</i>-fed animals resulted in significant attenuation of NF-κB activity following i.p. LPS administration in NF-κBlux^+/+ animals compared to mice that received T cells from placebo-fed controls (n = 6/group); (B) A subsequent study performed an identical adoptive transfer experiment except that nuclear NF-κB activation was measured using an ELISA system instead of biophotonic imaging - identical results were observed in that CD4⁺ T cells from <i>B. infantis</i>-fed animals significantly reduced LPS stimulated NF-κB activation (n = 5/group); (C) Removal of the CD25⁺ subpopulation from the CD4⁺ cells resulted in loss of the NF-kB suppressive activity while adoptive transfer of the CD25⁺ population alone replicated the CD4⁺ suppressive effect; (D) Similarily, the reduction in TNF-α secretion associated with CD4⁺ T cell transfer was lost when CD25⁺ were depleted but was replicated with transfer of CD25⁺ cells alone (n = 5/group). *p<0.05 versus placebo T cells; **p<0.05 versus CD4 T cells.</p

<i>Bifidobacterium infantis</i> consumption increases Treg numbers and activity in <i>S. typhimurium</i> infected mice.

<p>(A) There is a significant increase in the percentage of CD4⁺ cells co-expressing CD25 in the Peyer's patch of animals fed <i>B. infantis</i>, particularly following Salmonella infection; (B) There are also significantly more CD4⁺CD25⁺ cells in the spleen of animals fed <i>B. infantis</i>; (C) A representative flow cytometry dot-plot illustrates that the majority of the CD25⁺ cells within the spleen also stain positive for Foxp3 which is graphed for <i>B. infantis</i>-fed animals in (D); (E) CD4⁺CD25⁺ T cells suppressed proliferation of naïve CFSE labelled CD4⁺ cells while depletion of the CD25⁺ subset cells removed the suppressive effect. *p<0.05 versus placebo.</p

Dendritic cells are less activated in <i>B. infantis</i>-fed mice.

<p>(A) The percentage of CD11c⁺MHC II⁺ mature dendritic cells staining positive for the co-stimulatory molecule CD80 are reduced in the Peyer's patch of <i>B. infantis</i>-fed mice prior to and during <i>S. typhimurium</i> infection. (B) Splenocyte dendritic cell CD80 expression is similar in both groups of un-infected mice but is significantly up-regulated with Salmonella infection only in the placebo group. *p<0.05 versus placebo, n = 6–9 animals per group per timepoint.</p

Cytokine production is suppressed in <i>S. typhimurium</i>-infected mice when pre-treated with <i>B. infantis</i>.

<p>Cytokine release by isolated cells was examined immediately prior to <i>S. typhimurium</i> infection (Day 0) or four days (Day 4) after infection. (A) <i>In vitro</i> cytokine production by anti-CD3/CD28 stimulated Peyer's patch cells is significantly reduced in <i>S. typhimurium</i> infected mice when fed <i>B. infantis</i> with no differences being observed prior to infection; (B) <i>In vitro</i> IFN-γ and IL-10 production by anti-CD3/CD28 stimulated spleenocytes is significantly increased in placebo-fed animals following S. typhimurium infection. However, this increase was not observed in animals consuming <i>B. infantis</i>. TNF-α levels were similar for the two groups; (C) LPS stimulated splenocytes released comparable amounts of cytokine from the two groups of animals prior to infection but four days following Salmonella translocation, LPS stimulated TNF-α, IL-6 and MCP-1 release was significantly less in the <i>B. infantis</i>-fed animals. *p<0.05 versus placebo, n = 8/group at each timepoint.</p