Integrated analysis of gut metabolome, microbiome, and exfoliome data in an equine model of intestinal injury.

BACKGROUND: The equine gastrointestinal (GI) microbiome has been described in the context of various diseases. The observed changes, however, have not been linked to host function and therefore it remains unclear how specific changes in the microbiome alter cellular and molecular pathways within the GI tract. Further, non-invasive techniques to examine the host gene expression profile of the GI mucosa have been described in horses but not evaluated in response to interventions. Therefore, the objectives of our study were to (1) profile gene expression and metabolomic changes in an equine model of non-steroidal anti-inflammatory drug (NSAID)-induced intestinal inflammation and (2) apply computational data integration methods to examine host-microbiota interactions. METHODS: Twenty horses were randomly assigned to 1 of 2 groups (n = 10): control (placebo paste) or NSAID (phenylbutazone 4.4 mg/kg orally once daily for 9 days). Fecal samples were collected on days 0 and 10 and analyzed with respect to microbiota (16S rDNA gene sequencing), metabolomic (untargeted metabolites), and host exfoliated cell transcriptomic (exfoliome) changes. Data were analyzed and integrated using a variety of computational techniques, and underlying regulatory mechanisms were inferred from features that were commonly identified by all computational approaches. RESULTS: Phenylbutazone induced alterations in the microbiota, metabolome, and host transcriptome. Data integration identified correlation of specific bacterial genera with expression of several genes and metabolites that were linked to oxidative stress. Concomitant microbiota and metabolite changes resulted in the initiation of endoplasmic reticulum stress and unfolded protein response within the intestinal mucosa. CONCLUSIONS: Results of integrative analysis identified an important role for oxidative stress, and subsequent cell signaling responses, in a large animal model of GI inflammation. The computational approaches for combining non-invasive platforms for unbiased assessment of host GI responses (e.g., exfoliomics) with metabolomic and microbiota changes have broad application for the field of gastroenterology. Video Abstract.

Stimulation of embryo hatching and implantation by prostacyclin and peroxisome proliferator-activated receptor delta activation: implication in IVF.

BACKGROUND: Successful IVF depends in part on quality embryos. Recent work suggests that prostaglandin I(2) (PGI(2) or prostacyclin) promotes the development of embryos in vitro and enhances their implantation potential. The mechanism underlying the effects of PGI(2) is unclear. It has been reported that peroxisome proliferator-activated receptor delta (PPARdelta) mediates the effects of PGI(2) at the implantation sites. METHODS: The expression of PPARdelta in the preimplantation embryos was examined by RT-PCR, western blot analysis and immunohistochemistry. Synthetic PPARdelta ligand (L-165041) and PPARdelta targeted (PPARdelta(-/-)) embryos were used to reveal the roles of PPARdelta in PGI(2)-stimulated and spontaneous embryo development. RESULTS: Preimplantation embryos express PPARdelta, which is essential for the enhancing effect of PGI(2) and the spontaneous progression of preimplantation embryos. Enhanced blastocyst hatching by PGI(2) (P < 0.05) was abrogated by PPARdelta deletion. Blastocyst formation and embryo hatching were impaired in PPARdelta(-/-) embryos. PPARdelta deletion significantly reduced embryo cell proliferation (P < 0.01); PPARdelta activation increased embryo cell proliferation (P < 0.05). PPARdelta activation enhanced the implantation of wild-type (WT) embryos (P < 0.05); PPARdelta deletion reduced embryo implantation (P < 0.05). CONCLUSIONS: PPARdelta is essential for spontaneous and PGI(2)-stimulated embryo development and blastocyst hatching. The implantation of cultured embryos is enhanced by PPARdelta activation. PPARdelta represents a novel therapeutic target to improve IVF outcome.

Prostacyclin enhances the implantation and live birth potentials of mouse embryos.

BACKGROUND: Recently we reported that iloprost, a stable analogue of prostacyclin, enhanced mouse embryo hatching. Here we present a follow-up study to determine whether exposure to iloprost augments the implantation and live birth potentials of mouse embryos. METHODS: Two-cell embryos (C3B6F1) were harvested 42 h after HCG injection and cultured in medium supplemented with iloprost. After 48 h, the embryos were transferred to 2.5 day pseudopregnant gestational carriers. The number of gestation sacs was counted 72 h later; the number of live pups and the weight of pups and placentae were determined 14 days later. The implantation rate was defined as gestation sac per embryo transferred; the live birth rate was defined as live pup per embryo transferred. RESULTS: The prostacyclin analogue enhanced the implantation rate from 42 to 76% [relative risk 1.84, 95% confidence interval (CI) 1.38-2.43]. The rate of live pups also increased from 28 to 36% (relative risk 1.28, 95% CI 1.04-1.56). The weights of the pups and of the placentae of the two groups were comparable. CONCLUSION: Prostacyclin enhances the potentials of implantation and live birth of mouse embryos.

Prostacyclin enhances embryo hatching but not sperm motility.

BACKGROUND: Recently we discovered that the human oviduct synthesizes abundant prostacyclin (PGI(2)). Gene knock-out studies suggest that PGI(2) is essential to endometrial decidualization, but the effects of PGI(2) on sperm and embryos have not been reported. METHODS: The effects of PGI(2) on human sperm were analysed by a computer-assisted semen analysis system. The effects of PGI(2) on mouse embryos were examined based on the rates of complete hatching. The expression of PGI(2) receptor (IP) was evaluated by Western blot analysis and immunohistochemistry. The binding of PGI(2) to embryos was confirmed by radioligand binding assay. Finally, cAMP levels were assessed in PGI(2)-challenged embryos. RESULTS: Iloprost (a stable PGI(2) analogue) did not affect the motility or the overnight survivability of human sperm. Western blot analysis did not detect IP in the sperm plasma membrane. In contrast, the hatching of mouse embryos was enhanced by iloprost (ED(50) 6.7 nmol/l). Exposure to iloprost during 8-cell to morulae or morulae to early blastocyst stages was critical to enhanced hatching. This coincided with the developmental stage-specific expression of IP. Although iloprost bound to blastocysts, it did not significantly increase cAMP. CONCLUSION: PGI(2) enhanced the hatching of mouse embryos but not the motility of human sperm.

Preliminary analysis of azoxymethane-induced colon tumorigenesis in mouse aggregation chimeras.

Inbred mice exhibit differential susceptibility to colon carcinogens. The following study addresses the possibility that differences are intrinsic to colonic mucosa (cell autonomous) or are mediated by extracolonic systemic factors (e.g. liver activation of carcinogens). Our approach was to construct mouse aggregation chimeras, mice whose tissues are a mosaic of cells derived from two parental genotypes, from a susceptible (SWR) and a resistant (DBA/2) strain. Forty-five embryo aggregations yielded 11 viable pups, four of which were chimeric by coat color. Six-week-old SWR<-->BA/2 chimeras were injected i.p. with azoxymethane (AOM) once a week for 8 weeks (5 and 7.5 mg/kg body wt for 2 weeks followed by 10 mg/kg for 6 weeks) and tumor incidence in distal colon was evaluated 15 weeks after the last injection. Additional groups of parental mice received the same treatment. In the parental SWR treatment group, 1.7 +/- 0.82 tumors/colon were found. No tumors were observed in AOM-treated DBA/2 mice. In SWR<-->DBA/2 chimeras exposed to AOM, 2.8 +/- 2.1 tumors/colon were found. Tumor lineage was examined in paraffin sections stained with Dolichos biflorus agglutinin-peroxidase, a cell surface specific marker that stains intestinal endothelial cells of SWR and epithelial cells of DBA/2. Cellular lineage of tumors was further evaluated by microsatellite analysis of DNA isolated by microdissection. There was no significant difference in tumor incidence between SWR parental and chimera treatment groups. Histochemical analysis of tumor tissue in chimeras suggested that most tumors were derived from SWR. However, subsequent genetic analysis of tumors indicated mixed parental composition. These preliminary studies suggest that DBA/2 resistance mechanisms are not sufficient to protect adjacent SWR-derived epithelium from the tumorigenic effects of AOM.