Immunobiotic Bifidobacteria Strains Modulate Rotavirus Immune Response in Porcine Intestinal Epitheliocytes via Pattern Recognition Receptor Signaling

In this work, we aimed to characterize the antiviral response of an originally established porcine intestinal epithelial cell line (PIE cells) by evaluating the molecular innate immune response to rotavirus (RVs). In addition, we aimed to select immunomodulatory bacteria with antiviral capabilities. PIE cells were inoculated with RVs isolated from different host species and the infective titers and the molecular innate immune response were evaluated. In addition, the protection against RVs infection and the modulation of immune response by different lactic acid bacteria (LAB) strains was studied. The RVs strains OSU (porcine) and UK (bovine) effectively infected PIE cells. Our results also showed that RVs infection in PIE cells triggered TLR3-, RIG-I- and MDA-5-mediated immune responses with activation of IRF3 and NF-κB, induction of IFN-β and up-regulation of the interferon stimulated genes MxA and RNase L. Among the LAB strains tested, Bifidobacterium infantis MCC12 and B. breve MCC1274 significantly reduced RVs titers in infected PIE cells. The beneficial effects of both bifidobacteria were associated with reduction of A20 expression, and improvements of IRF-3 activation, IFN-β production, and MxA and RNase L expressions. These results indicate the value of PIE cells for studying RVs molecular innate immune response in pigs and for the selection of beneficial bacteria with antiviral capabilities.     

Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression

Background

We previously showed that evaluation of anti-inflammatory activities of lactic acid bacteria in porcine intestinal epithelial (PIE) cells is useful for selecting potentially immunobiotic strains.

Objective

The aims of the present study were: i) to select potentially immunomodulatory bifidobacteria that beneficially modulate the Toll-like receptor (TLR)-4-triggered inflammatory response in PIE cells and; ii) to gain insight into the molecular mechanisms involved in the anti-inflammatory effect of immunobiotics by evaluating the role of TLR2 and TLR negative regulators in the modulation of proinflammatory cytokine production and activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways in PIE cells.

Results

Bifidobacteria longum BB536 and B. breve M-16V strains significantly downregulated levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and IL-6 in PIE cells challenged with heat-killed enterotoxigenic Escherichia coli. Moreover, BB536 and M-16V strains attenuated the proinflammatory response by modulating the NF-κB and MAPK pathways. In addition, our findings provide evidence for a key role for the ubiquitin-editing enzyme A20 in the anti-inflammatory effect of immunobiotic bifidobacteria in PIE cells.

Conclusions

We show new data regarding the mechanism involved in the anti-inflammatory effect of immunobiotics. Several strains with immunoregulatory capabilities used a common mechanism to induce tolerance in PIE cells. Immunoregulatory strains interacted with TLR2, upregulated the expression of A20 in PIE cells, and beneficially modulated the subsequent TLR4 activation by reducing the activation of MAPK and NF-κB pathways and the production of proinflammatory cytokines. We also show that the combination of TLR2 activation and A20 induction can be used as biomarkers to screen and select potential immunoregulatory bifidobacteria strains.     

Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.     

Functional characteristics of Peyer's patch lymphoid cells. IV. Effect of antigen feeding on the frequency of antigen-specific B cells.

The frequency of B cells in Peyer's patches from normal BDF(1) and sheep red blood cell (SRBC)-fed BDF(1) mice that could respond to antigenic determinants on SRBC and trinitrophenyl (TNP) was determined using an in vitro system of limiting dilution analysis. In normal mice, one B cell in 1.9 x 10(4) Peyer's patch cells could be induced to an anti-SRBC response and one B cell in 3.6 x 10(4) Peyer's patch cells could be induced to an anti-TNP response. The frequency of B cells capable of responding to SRBC in normal mice was similar in Peyer's patches and spleen. However, after feeding mice SRBC for 3 weeks, there was a 6-fold reduction in the frequency of B cells in Peyer's patches capable of responding to SRBC but no change in the frequency of B cells capable of responding to TNP. The average clone size of Peyer's patch B cells responding to SRBC was similar in normal and SRBC-fed mice. Although antigen-feeding does not stimulate Peyer's patch B cells in situ to humoral antibody synthesis, antigen-feeding can markedly alter the reactivity of the antigen-sensitive cell population in Peyer's patches. We previously demonstrated that T cells in Peyer's patches could be specifically carrier primed for helper function by SRBC feeding. We have now demonstrated that antigen-feeding reduced significantly the frequency of B cells in Peyer's patches capable of responding to the fed antigen. Peyer's patches appear to serve an important function as a sampling site for intestinal antigens.     

Toll-like receptor 4 and cytokine expression involved in functional immune response in an originally established porcine intestinal epitheliocyte cell line

To study the immune responses of porcine intestinal epithelial cells to gram-negative bacteria via toll-like receptors (TLRs), originally established porcine intestinal epitheliocyte (PIE) cells were treated with lipopolysaccharide (LPS) or swine-specific enterotoxigenic Escherichia coli (ETEC). Real-time quantitative PCR revealed that PIE cells expressed TLR1-9 and MD-2 mRNAs, preferentially expressed TLR4/MD-2. Immunostaining of PIE cells revealed that TLR4 was precisely expressed in PIE cells at the protein level. PIE cells treated with LPS had up-regulated expression of several TLRs (TLR2, 3, 4, 5 and 8), type 1 helper T (Th1) cytokines (interleukin (IL)-1alpha, IL-1beta, IL-6, IL-15, 18, leukemia inhibitory factor (LIF), and interferon (IFN)-beta), and chemokines (monocyte chemoattractant protein (MCP)-1 and IL-8). ETEC enhanced the expression of TLR2, Th1 type cytokines (IL-1alpha, IL-12p35 and IL-6) and chemokines (MCP-1 and IL-8). These results indicate that PIE induces inflammatory responses by up-regulating Th1 cytokines and chemokines in response to LPS or ETEC, suggesting that PIE is a useful cell line for studying inflammatory responses via TLR4/MD-2 in intestinal epithelial cells.     

The correlation between surface hydrophobicity and adherence of Bifidobacterium strains from centenarians' faeces

To develop food-grade bifidobacteria micro-ecologics, screening for Bifidobacteria strains which can adhere to intestinal epithelial cells was finished. Twenty-three bifidobacterial strains tested were isolated from centenarians in Bama country, the fifth long-lived district in the world. Surface hydrophobicity and adherence capability to intestinal epithelial cells in vitro of bifidobacteria were simultaneously investigated for the first time. It has been demonstrated that all the strains exhibited adhesive properties to some extent using intestinal Caco-2 cell line in in vitro model. It could be conclude that the higher hydrophobic strains the stronger adhesive capability. The highest value of hydrophobicity (37.24+/-1.45% and 32.06+/-1.21%) was obtained for strains H-10 and I-6, respectively; correspondingly, the strongest adherence ability (49.47+/-4.88/cell and 47.33+/-2.72/cell) was achieved, respectively. Correlation between surface hydrophobicity and adherence ability of different Bifidobacterium strains including polynomial regression equation (R2=0.78) had been achieved. The present study provided a liable and effective method for screening bifidobacteria with the ability to adhere to intestinal epithelial cells.     

Screening of bifidobacteria with acquired tolerance to human gastrointestinal tract

Tolerance capabilities of 38 Bifidobacterium strains were achieved by simulating the micro-environment of human gastrointestinal tract using modified MRSC broth (pH 3.0). Fourteen strains of them with high viability were obtained in MRSC with 0.3% bile salts. Sequently, six strains of bifidobacteria with higher survivability were picked in MRSC broth (pH 3.0) supplemented with 1-20mg/ml bile salt. Finally, strain A04 with the optimal ability was chosen for further studies. It had been seen that Bifidobacterium breve A04 had better survival capability to 0.5% pepsin (w/v) or 1% pancreatin (w/v) than other bifidobacteria, and viable bacteria were above 8.00 log cfu/ml after incubation for 24h. Meanwhile, it had higher adhesive capability to HT-29 cells in vitro and average adhesive bacteria numbers reached 12.8+/-0.9 for each HT-29 cell. The results indicated that the ability to tolerate gastroenteric environment and the adhesive capacity to HT-29 cells among Bifidobacterium strains was different. B. breve A04 has several aspects of advantages and may be regarded as potential probiotics.     

Increased resistance of mice to Salmonella enterica serovar Typhimurium infection by synbiotic administration of Bifidobacteria and transgalactosylated oligosaccharides

AIMS: The anti-infectious activity of Bifidobacteria in combination with transgalactosylated oligosaccharides (TOS) against Salmonella enterica serovar Typhimurium LT-2 in an opportunistic antibiotic-induced murine infection model in mice was examined. METHODS AND RESULTS: B. breve (strain Yakult) with natural resistance to streptomycin sulphate (SM, MIC: > 4 mg ml(-1)), when given daily at a dose of 108 cfu/mouse orally under SM treatment was constantly excreted at 10(10) cfu g(-1) faeces so long as SM was administered, even at 2 weeks after discontinuing administration of B. breve. Explosive intestinal growth and subsequent extra-intestinal translocation of orally infected LT-2 under SM treatment were inhibited by B. breve colonization, and this anti-infectious activity was strengthened by synbiotic administration of TOS with B. breve. Comparison of anti-Salmonella activity among several Bifidobacterium strains with natural resistance to SM revealed that strains such as B. bifidum ATCC 15696 and B. catenulatum ATCC 27539T conferred no activity, even when they reached high population levels similar those of effective strains such as strain Yakult and B. pseudocatenulatum DSM 20439. Both the increase in the concentration of organic acids and the lowered pH in the intestine due to bifidobacterial colonization correlated with the anti-infectious activity. Moreover, the crude cecal extract of B. breve-colonized mice exerted growth-inhibitory activity against LT-2 in vitro, whereas that of the ineffective B. bifidum-colonized cecum showed much lower activity. CONCLUSIONS: Intestinal colonization by bifidobacteria given exogenously together with TOS during antibiotic treatment prevents the antibiotic-induced disruption of colonization resistance to oral infection with S. enterica serovar Typhimurium, and the metabolic activity needed to produce organic acids and lower the intestinal pH is important in the anti-infectious activity of synbiotics against enteric infection with Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that certain bifidobacteria together with prebiotics may be used for the prophylaxis against opportunistic intestinal infections with antibiotic-resistant pathogens.     

Effects of antigen-feeding on intestinal and systemic immune responses. I. priming of precursor cytotoxic T cells by antigen feeding.

The ability of antigens administered by the intestinal route to alter the cytotoxic T lymphocyte (CTL) population in intestinal and extraintestinal sites was examined by feeding mice tumor cells bearing H-2 or non-H-2 gene-coded cell surface alloantigenic differences. Peyer's patches from mice fed tumor cells differing at H-2 demonstrated a 12-fold increase in specific CTL activity over controls after stimulation of culture but the precursor CTL population in Peyer's patches was not expanded by feeding mice cells bearing minor H gene coded alloantigenic differences. In contrast, the precursor CTL pool in spleen was increased both in mice fed cells bearing alloantigenic differences coded for by H-2 and non-H-2 gene loci. The precursor CTL population in mesenteric lymph nodes (MN) was not expanded significantly by tumor cell feeding. Feeding was nearly as effective as i.p. priming at expanding the Peyer's patch precursor CTL population when tumor cells bearing H-2 differences were used, whereas i.p. priming was more effective than feeding in expanding the precursor CTL population in spleen.     

Probiotic characteristics and in vitro compatibility of a combination of Bifidobacterium breve M-16 V,Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536

The consumption of probiotic-based products has risen greatly in recent decades. Due to their probiotic characteristics, microorganisms such as lactobacilli and bifidobacteria are in daily use in the production of food supplements. In the present study, three bifidobacterial strains (Bifidobacterium breve M-16 V, Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536) were tested for growth compatibility, resistance to antimicrobial agents, antibacterial activity against pathogens, resistance to gastric acidity, bile salt hydrolysis and adhesion to the human intestinal epithelial cell line HT29. All of these strains were resistant to gentamycin, but none showed in vitro growth incompatibility or the presence of known resistance determinants. B. breve M-16 V had the best probiotic characteristics and, indeed, was the only strain possessing antibacterial activity against Escherichia coli and Klebsiella pneumoniae. All strains were resistant to simulated gastric juice, while only B. longum subsp. longum BB536 and B. breve M-16 V showed a bile salt hydrolytic activity. Interestingly, a strong adhesion to HT29 cells was observed in all Bifidobacterium strains. In conclusion, B. breve M-16 V, B. longum subsp. longum BB536 and B. longum subsp. infantis M-63 showed several promising characteristics as probiotic strains.     

Generation and characterization of monoclonal antibodies recognizing follicle epithelial M cells in rabbit gut-associated lymphoid tissues

A panel of mouse B cell hybridomas producing monoclonal antibodies (mAb) directed against rabbit M cell-containing epithelia was developed. By immunohistochemistry, the mAb 5D9, 5B11, 1D9, and 4G2 were found to label approximately 50% of the follicle-associated epithelial (FAE) cell populations overlying lymphoid follicles in Peyer's patches, cecal patch, sacculus rotundus, and appendix. The cell staining was localized to FAE cell basolateral surfaces outlining the M cell pockets which enclosed clusters of mononuclear leukocytes, and extended from the crypts of Peyer's patches and sacculus rotundus, and appendiceal crevices, to the apices of domes. In contrast, the stem cell and proliferative regions facing the lamina propria were devoid of immunologically reactive sites. The mAb 5D9, 1D9, and 4G2 did not recognize antigens associated with non-FAE cells in the intestinal lymphoid tissues examined. Only the mAb 5B11 labeled apical surfaces of Peyer's patch and cecal patch non-FAE. However, this mAb did not label interdomal colonic epithelial cells in sacculus rotundus and appendix. Besides recognizing FAE cells, the mAb 4G2 recognized a cross-reactive antigen displayed by dome and lymphoid follicle lymphocytes. By flow cytometry, the mAb 5D9, 5B11, and 1D9 were shown to stain from 14 to 29% of the cells in M cell-enriched populations prepared from Peyer's patches, sacculus rotundus, and appendix, whereas mAb 4G2 was found to recognize 44-54% of the cells. Two-color flow cytometric analysis showed that the mAb stained a functionally distinct subpopulation of Peyer's patch phagocytic cells and did not recognize spleen macrophages. These findings indicate that the panel of mAb recognized novel antigens expressed by FAE cells overlying intestinal lymphoid aggregates, and that the mAb allow identification of phagocytic M cells in suspensions of FAE cells.     

Stimulation of the secretion of pro-inflammatory cytokines by Bifidobacterium strains

To characterize the ability of bifidobacteria to affect the production of macrophage-derived cytokines, a murine macrophage-like cell line, J774.1, was cultured in the presence of 27 strains of heat-inactivated bifidobacteria. Bifidobacterium adolescentis and B. longum, known as adult-type bifidobacteria, induced significantly more pro-inflammatory cytokine secretion, IL-12 and TNF-alpha, by J774.1 cells, than did the infant-type bifidobacteria, B. bifidum, B. breve, and B. infantis (P<0.01). In contrast, B. adolescentis did not stimulate the production of anti-inflammatory IL-10 from J774.1 cells as the other tested bacteria did. The results suggest that the adult-type bifidobacteria, especially B. adolescentis, may be more potent to amplify but less able to down-regulate the inflammatory response.     

Cytokeratin 18 is an M-cell marker in porcine Peyer's patches

The intermediate filaments of the dome epithelium of porcine Peyer's patches were studied by immunohistochemistry. The labelling patterns of monospecific antibodies directed against cytokeratins 8, 18 and 19 differed considerably. About 40% of the dome epithelial cells were intensely labelled by three different anti-cytokeratin 18 antibodies, indicating that large amounts of cytokeratin 18 are present in these cells. In order to verify that these cytokeratin-18-immunoreactive cells were M-cells, uptake studies using fluorescein-labelled yeast particles were performed. Numerous yeast particles were found exclusively in dome epithelial cells that were highly positive for cytokeratin 18, thus representing M-cells. In contrast, the content of cytokeratin 19 in M-cells was lower than that in neighbouring enterocytes. The labelling intensity of cytokeratin 8 did not differ between M-cells and enterocytes. In addition, the absence of vimentin and desmin from the dome epithelium of porcine Peyer's patches was demonstrated. The results show (1) that porcine M-cells differ from enterocytes in the composition of their cytoskeleton, (2) that cytokeratin 18 is a useful marker for detecting porcine M-cells and (3) that this marker directly correlas with M-cell function.     

The vaginal Bifidobacterium flora in women of reproductive age

The composition of vaginal bifidoflora in 56 clinically healthy women of reproductive age was studied. The study revealed that four species of bifidobacteria, viz. Bifidobacterium bifidum, B. breve, B. adolescentis 2 and B. longum, dominated in the composition of this bifidobacterial population. Nine out of 11 isolated strains were found to be capable of inhibiting indicator microorganisms Staphylococcus aureus and Enterococcus faecalis when tested in vitro; in addition, strains B. adolescentis 2 F1, B. bifidum G1, B. breve P2 and B. longum Z4 inhibited Klebsiella ozaenae, Pseudomonas aeruginosa, Escherichia coli and were also active acid producers. Three of these 4 bifidobacterial strains were capable of adhesion to vaginal epitheliocytes, while B. bifidum G1 was practically incapable of adherence to these cells, similarly to B. bifidum strain 791 of intestinal origin. In addition, the spectra of antibiotic susceptibility varied from strain to strain, but all bifidobacterial strains were susceptible to benzylpenicillin and resistant to lomefloxacin, most of them being also resistant to cyprofloxacin and gentamicin. Thus the data presented in this work are indicative of the possibility and advantages of using bifidobacterial strains belonging to this ecological niche as probiotics for the correction of the microflora of the urogenital tract in females.     

Response of Peyer's patch lymphocyte subsets to Giardia muris infection in BALB/c mice. I. T-cell subsets

Initial cellular events in the intestinal immune response occur within Peyer's patches and are subject to complex regulation by T cells. The aim of this study was to analyze the response of murine Peyer's patch T, T-helper (Th), and T-suppressor (Ts) cells to Giardia muris infection. Immunocompetent BALB/c mice were infected with G. muris cysts and, at serial times during the infection, Peyer's patch leukocyte suspensions were incubated with fluorescent monoclonal antibodies that identified murine leukocytes, T, Th, or Ts lymphocytes. These suspensions were examined by flow cytometry to quantify each T-cell subset as a percentage of total leukocytes. Total Peyer's patch leukocytes more than doubled in number during the course of G. muris infection and returned to control levels as the infection was cleared. The percentages of Peyer's patch Th and Ts lymphocytes were 34.1 +/- 0.8% (mean +/- SEM) and 6.2 +/- 0.3%, respectively, in the absence of infection, and did not change significantly during Giardia infection. The Th/Ts ratio in Peyer's patches was 5.6 +/- 0.2 in uninfected BALB/c mice and also did not change significantly during clearance of G. muris. We conclude that Peyer's patch leukocytes double in number in response to G. muris infection in immunocompetent mice, G. muris infection does not lead to altered percentages of Peyer's patch T, Th, or Ts lymphocytes, and clearance of G. muris infection is associated with a Peyer's patch Th/Ts ratio of greater than 5.     

Uptake and transport of copolymer biodegradable microspheres by rabbit Peyer's patch M cells

In this study, we demonstrate the role of M cells in uptake of poly(D-L-lactic-co-glycolic acid) (PLGA) microspheres and transport into rabbit Peyer's patches. Microspheres 1 to 10 m in diameter composed of 50:50 lactic acid:glycolic acid were instilled into in-testinal segments containing jejunal or ileal Peyer's patches, and uptake by M cells was examined by electron microscopy. PLGA microspheres visualized as electron-lucent, spherical particles were taken up by M cells by pseudopod-like extensions of the M cell apical membrane and translocated to the pocket region containing mononuclear leukocytes within 60 min. These results indicate that PLGA microspheres can be directed to M cell apical surfaces for delivery to immunocompetent cells in gut-associated lymphoid tissues.     

Interaction of vasoactive intestinal peptide with mouse lymphocytes: specific binding and the modulation of mitogen responses

Binding of radioiodinated vasoactive intestinal peptide (VIP) to mouse lymphocytes has been investigated. Specific cell binding of 125I-VIP was demonstrated with lymphocytes from mesenteric lymph nodes, subcutaneous lymph nodes, spleen, and Peyer's patches. The binding of VIP by these cells was accounted for by VIP binding sites upon T cells rather than non-T cells. In the presence of VIP, the in vitro response of lymphocytes to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) was inhibited in a dose-dependent fashion, whereas that to the B cell mitogen lipopolysaccharide (LPS) was not. There was a close correlation between the potency of VIP and some structurally related peptides for inhibition of 125I-VIP binding and the effect of those peptides on T cell mitogen responses. These observations demonstrate that mouse T lymphocytes have specific VIP receptors and that VIP can modulate the response of T cells to mitogenic stimulation. VIP may be an important immunoregulatory molecule, and may be implicated in the regulation of T cell function in mucosal tissues innervated by VIP-containing neurons.     

B1 cells contribute to serum IgM,but not to intestinal IgA,production in gnotobiotic Ig allotype chimeric mice

B1 cells are a significant source of natural serum IgM, thereby serving as a first line of defense against systemic bacterial and viral infections. They can migrate to the intestinal lamina propria and differentiate into IgA-producing plasma cells and thus might play a similar role in mucosal immunity. To investigate the contribution of B1 cells to the intestinal IgA response induced by the commensal flora in immunocompetent animals, we generated gnotobiotic and conventionally reared Ig allotype chimeric mice. In this system B1- and B2-derived Abs can be distinguished based on different allotypes. FACS analysis of peritoneal cavity cells and analysis of B1- and B2-derived serum IgM indicated stable B1/B2 chimerism and the establishment of a functional B1 population. Monoassociation with either Morganella morganii, Bacteroides distasonis, or segmented filamentous bacteria induced germinal center reactions in Peyer's patches and led to the production of intestinal IgA, partially reactive with bacterial Ag. A considerable amount of serum IgM was B1 cell derived in both monoassociated and conventionally reared mice. However, most of the total as well as bacteria-specific intestinal IgA was produced by B2 cells. These data suggest that intestinal IgA production induced by commensal bacteria is mainly performed by B2, not B1, cells.     

Gene expression profiling of jejunal Peyer’s patches in juvenile and adult pigs

Peyer's patches are organized lymphoid tissues of the small intestine that play a critical role in disease resistance and oral tolerance. Peyer's patches in the jejunum contain lymphocytes, dendritic cells, macrophages, villous epithelium, and specialized follicle-associated epithelium. Little is known about the mechanisms and processes by which cells of the Peyer's patches discriminate food nutrients and commensal microflora from pathogenic microbiota. We hypothesize that the jejunal Peyer's patches express genes that mediate and regulate its essential functions. Expression patterns of approximately 2600 cDNAs from a porcine Peyer's patch subtracted library were examined by microarray profiling. Individual mRNAs of interest were further examined by quantitative RT-PCR. Innate immunity-associated genes, including complement 3 and lysozyme, and the genes for epithelial chloride channel and trappin 1 were highly expressed by jejunal Peyer's patch in both juvenile and adult pigs. The growth- and apoptosis-associated genes CIDE-B, GW112, and PSP/Reg I (pancreatic stone protein or regenerating gene) were differentially expressed in juvenile pig Peyer's patches. Many sequences which were highly expressed in jejunal Peyer's patches have previously been described with functions in epithelial cells. Animal-to-animal variation in basal jejunal Peyer's patch gene expression was considerable and reflects the dynamic physiological environment of the gut in addition to genetic, epigenetic, and microbiological variation in the small intestine.