Transciptome profiling of small intestinal epithelial crypt and villi in germfree versus conventional neonatal piglets. Sus scrofa

To gain insight into host-microbe interactions in a piglet model, a functional genomics approach has been applied to address the working hypothesis that transcriptionally regulated genes associated with promoting epithelial barrier function evolved as a defensive response to the intestinal microbiota. Cesarean-derived germfree (GF) newborn piglets were colonized with adult swine feces, and villus and crypt epithelial cell transcriptomes from colonized and GF neonatal piglets were compared using laser-capture microdissection and high-density porcine oligonucleotide microarray technology. Consistent with our hypothesis, results revealed that resident microbiota induces the expression of genes encoding proteins involved in promoting intestinal epithelial cell turnover and mucus biosynthesis and priming of the immune system. Furthermore, differential expression of genes associated with antigen presentation (SLA-7, B2M, TAP1 and TAPBP) demonstrates that microbiota induced immune responses using a distinct regulatory mechanism common for these genes. Intestinal microbiota stimulated interferon (IFN) receptor-mediated signaling cascade using signal transducer and activator of transcription-1 (STAT1), STAT2 and IFN regulatory factor-7 transcription factors to activate IFN-inducible genes. In addition, excessive inflammatory responses were prevented by activating RNA expression of inhibitory-kappa-B and Toll-interacting protein, along with downregulating the expression of a gene encoding GATA binding protein-1 consistent with the maintenance of intestinal homeostasis. This study supports the concept that the intestinal epithelium has evolved to maintain a physiological state of inflammation with respect to continuous microbial exposure, which serves to maintain tight intestinal barrier but prevent overt inflammatory responses that would compromise barrier function. Keywords: oligonucleotide array Overall design: Four piglets from one isolator were maintained germfree and four piglets in other isolator were conventionalized by fecal slurry. Laser capture microdissection was used to recover epithelial villus and crypt cells from frozen ileal sections. Porcine oligonucleotide set represents 13,297 porcine cDNAs and ESTs. A reference design was used in which each of the experimental samples was cohybridized with the reference sample that contained equal amounts of all the RNA samples used in the experiment, which allowed the treatment of fluorescence ratios as measurements of relative expression.