Gut microbiota-mediated bile acid transformations alter the cellular response to multidrug resistant transporter substrates in vitro: focus on P-glycoprotein

Pharmacokinetic research at the host-microbe interface has been primarily directed toward effects on drug metabolism, with fewer investigations considering the absorption process. We previously demonstrated that the transcriptional expression of genes encoding intestinal transporters involved in lipid translocation are altered in germ-free and conventionalized mice possessing distinct bile acid signatures. It was consequently hypothesized that microbial bile acid metabolism, which is the deconjugation and dehydroxylation of the bile acid steroid nucleus by gut bacteria, may impact upon drug transporter expression and/or activity and potentially alter drug disposition. Using a panel of three human intestinal cell lines (Caco-2, T84, and HT-29) that differ in basal transporter expression level, bile acid conjugation-, and hydroxylation-status was shown to influence the transcription of genes encoding several major influx and efflux transporter proteins. We further investigated if these effects on transporter mRNA would translate to altered drug disposition and activity. The results demonstrated that the conjugation and hydroxylation status of the bile acid steroid nucleus can influence the cellular response to multidrug resistance (MDR) substrates, a finding that did not directly correlate with directionality of gene or protein expression. In particular, we noted that the cytotoxicity of cyclosporine A was significantly augmented in the presence of the unconjugated bile acids deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) in P-gp positive cell lines, as compared to their taurine/glycine-conjugated counterparts, implicating P-gp in the molecular response. Overall this work identifies a novel mechanism by which gut microbial metabolites may influence drug accumulation and suggests a potential role for the microbial bile acid-deconjugating enzyme bile salt hydrolase (BSH) in ameliorating multidrug resistance through the generation of bile acid species with the capacity to access and inhibit P-gp ATPase. The physicochemical property of nonionization is suggested to underpin the preferential ability of unconjugated bile acids to attenuate the efflux of P-gp substrates and to sensitize tumorigenic cells to cytotoxic therapeutics in vitro. This work provides new impetus to investigate whether perturbation of the gut microbiota, and thereby the bile acid component of the intestinal metabolome, could alter drug pharmacokinetics in vivo. These findings may additionally contribute to the development of less toxic P-gp modulators, which could overcome MDR

Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine

Unconjugated Bile Acids Influence Expression of Circadian Genes: A Potential Mechanism for Microbe-Host Crosstalk.

Disruptions to circadian rhythm in mice and humans have been associated with an increased risk of obesity and metabolic syndrome. The gut microbiota is known to be essential for the maintenance of circadian rhythm in the host suggesting a role for microbe-host interactions in the regulation of the peripheral circadian clock. Previous work suggested a role for gut bacterial bile salt hydrolase (BSH) activity in the regulation of host circadian gene expression. Here we demonstrate that unconjugated bile acids, known to be generated through the BSH activity of the gut microbiota, are potentially chronobiological regulators of host circadian gene expression. We utilised a synchronised Caco-2 epithelial colorectal cell model and demonstrated that unconjugated bile acids, but not the equivalent tauro-conjugated bile salts, enhance the expression levels of genes involved in circadian rhythm. In addition oral administration of mice with unconjugated bile acids significantly altered expression levels of circadian clock genes in the ileum and colon as well as the liver with significant changes to expression of hepatic regulators of circadian rhythm (including Dbp) and associated genes (Per2, Per3 and Cry2). The data demonstrate a potential mechanism for microbe-host crosstalk that significantly impacts upon host circadian gene expression

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 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

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