ABSTRACT: Although immune mechanisms have been described for Peyer’s patch function, in discriminating food nutrients and commensal microflora from pathogenic challenge, an understanding of gene expression patterns associated with normal intestinal homeostasis is lacking. The effects of commensal bacterial colonization on global gene expression in the small intestine of germ-free mice have been described (1, 2). However, no studies have compared expression patterns of various healthy lymphoid tissues in conventionally-raised pigs or other species. The objective of this study was to investigate the pattern of normal gene expression in jejunal Peyer’s patches (JPP) of healthy pigs. We hypothesized that gene expression of intact jejunal Peyer's patch is characterized by genes associated with absorption and secretory functions as well as genes associated with GALT-specific immune functions. Eight hybridizations were performed, with dye swaps of JPP mRNA from four juvenile pigs. Each hybridization compared JPP from an individual pig to the jejunal mesenteric lymph node reference sample, which was pooled from all juvenile pigs in the study. However, two of the hybridizations resulted in poor quality data, and were excluded from further analysis. Normalization, hierarchical clustering, data visualization, and statistical analysis were performed by GeneSpring version 6.2 (Silicon Genetics, Redwood, CA). GeneSpring standardized the intensities for each spot by subtracting the local background, and then normalized globally by locally weighted linear regression (Lowess). A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. Spots with a flag value of “0” were excluded. The intensity of negative control DNA-containing spots was used to additionally exclude low intensity data. Negative controls include the gene for Arabidopsis thaliana homeodomain-like protein (AY054571); 200 ng/microl Poly(dA) (Amersham, Piscataway, NJ); a 461 bp fragment of the kanamycin-resistant gene, amplified from the pET24b+ plasmid (Novagen, Madison, WI); PRRS virus strain VR-2332 (PRU87392) ORFs 2, 3, 4, 5, 6 and 7; pCMVSport 6 plasmid (Invitrogen, Carlsbad, CA); and pGEM-T plasmid (Promega, Madison WI). The mean intensity of the negative controls for both Cy3 and Cy5 was calculated for each slide, and spots with a mean intensity less than the mean intensity + 2SD were excluded from further analysis. After normalization, GeneSpring averaged replicate spots for each clone across hybridizations. GeneSpring used the Cross Gene Error Model, which was based on replicate values. A Student’s t-test with the Benjamini and Hochberg false discovery rate multiple testing correction (MTC) verified the difference between the natural log of the normalized gene expression ratio (JPP:j-MLN) and a ratio of 1.0. p-values less than 0.05 were considered significant. Series_references: 1.Fukushima K, Ogawa H, Takahashi K, Naito H, Funayama Y, Kitayama T, Yonezawa H, and Sasaki I. Non-pathogenic bacteria modulate colonic epithelial gene expression in germ-free mice. Scand J Gastroenterol 38: 626-634, 2003. 2. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, and Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291: 881-884, 2001.
