Swine Intestinal Immunity via Toll-like Receptors and Its Advanced Application to Food Immunology

Recent interest has focused on the importance of intestinal immunity for the host defense, but to date, not much has been known about the underlying mechanisms. Toll-like receptor (TLR) family plays an important role in the defense through recognizing bacterial pathogen associated molecular patterns (PAMPs). Our research on the bioregulatory function of food products has investigated the immunoregulatory effects of lactic acid bacteria (LAB) via TLRs. Studies in swine, which is expected as a human model, have been examined intestinal immunoregulation by the LAB. Our research has now demonstrated modulation of intestinal immunity mediated by TLRs in Peyer\u27s patches and the mesenteric lymph nodes. On the basis of our study, efforts have also been made to develop an immunoassay system for immunobiotic LAB DNA and cell wall components to evaluate immunoregulation by the LAB via TLRs. The findings in our research activities may provide important clues at the molecular level on TLR signal transduction pathways and recognition mechanisms. They also provide impetus to further delineate the activation mechanism of the innate immune response. In addition, identification of biofactors from LAB with immunoactivity, and better understanding of cytokine induction and intestinal immune regulation hold promise in basic research and development of "immunobiotic foods" to prevent specific diseases

Variation of Rumen Bacterial Diversity in Steers after the Beginning of Grazing

Holstein heifers before or after puberty often are herded on public pastures in Japan. The herbage intake and rumen fermentation of grazing heifers that are not adapted for fresh herbage decreases due to a change of feed from stall-fed dried forage to fresh herbage. This limits their performance during the first several weeks on pasture. Thus, the feeding program such as supplementation before and after the beginning of grazing is important. An increase in ammonia concentration and a decline in fibre degradation in the rumen of a heifer (both of which occur simultaneously with low herbage intake and rumen fermentation) would be caused by the reduced capacity of various bacteria to produce peptides and degrade fibre (Oshio and Tahata 1981). This suggests that variation in rumen bacterial diversity plays an important role in herbage intake and rumen fermentation. However, less information is available on how bacterial diversity in heifers varies during the first few weeks of grazing. This information will provide the basis for designing nutritional management programs for heifers before and after the beginning of grazing. The objective of this study was to determine how herbage intake, digestibility, and rumen bacterial diversity vary in steers that have started grazing without adaptation for fresh herbage during the first 4 weeks after the beginning of grazing

Identification of a potent immunostimulatory oligodeoxynucleotide from Streptococcus thermophilus lacZ

Immunostimulatory sequences of oligodeoxynucleotides (ODNs), such as CpG ODNs, are potent stimulators of innate immunity. Here, we identified a strong immunostimulatory CpG ODN, which we named MsST, from the lac Z gene of Streptococcus (S.) thermophilus ATCC19258, and we evaluated its immune functions. In in vitro studies, MsST had a similar ability as the murine prototype CpG ODN 1555 to induce inflammatory cytokine production and cell proliferation. In mouse splenocytes, MsST increased the number of CD80+CD11c+and CD86+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells. We also analyzed the effects of MsST on the expression of regulatory cytokines by real-time quantitative PCR. MsST was more potent at inducing interleukin-10 expression than the ODN control 1612, indicating that MsST can augment the regulatory T cell response via Toll-like receptor 9, which plays an important role in suppressing T helper type 2 responses. These results suggest that S. thermophilus, whose genes include a strong Immunostimulatory sequence-ODN, is a good candidate for a starter culture to develop new physiologically functional foods and feeds.ArticleANIMAL SCIENCE JOURNAL. 80(5):597-604 (2009)journal articl

CpG oligodeoxynucleotides induce strong up-regulation of interleukin 33 via Toll-like receptor 9

We previously reported the strong immunostimulatory effects of a CpG oligodeoxynucleotide (ODN), designated MsST, from the lacZ gene of Streptococcus (S.) thermophilus ATCC19258. Here we show that 24 h of stimulation with MsST in mouse splenocytes and peritoneal macrophages strongly induces expression of interleukin (IL)-33, a cytokine in the IL-1 superfamily. Other IL-1 superfamily members, including IL-1 alpha, IL-1 beta and IL-18, are down-regulated after 24 h of stimulation of MsST. We also found that MsST-induced IL-33 mRNA expression is inhibited by the suppressive ODN A151, which can inhibit Toll-like receptor 9 (TLR9)-mediated responses. This is the first report to show that IL-33 can be induced by CpG ODNs. The strong induction of IL-33 by MsST suggests that it may be a potential therapeutic ODN for the treatment of inflammatory disease. The presence of a strong CpG ODN in S. thermophilus also suggests that the bacterium may be a good candidate as a starter culture for the development of new physiologically functional foods.ArticleBIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 394(1):81-86 (2010)journal articl

Immunobiotic Lactobacillus jensenii as immune-health promoting factor to improve growth performance and productivity in post-weaning pigs

Background: Immunoregulatory probiotics (immunobiotics) have been proposed to improve piglets' immune system to avoid intestinal infections and reduce unproductive inflammation after weaning. Previously, it was demonstrated that Lactobacillus jensenii TL2937 (LjTL2937) attenuated the inflammatory response triggered by activation of Toll-like receptor 4 (TLR-4) in porcine intestinal epithelial (PIE) cells and antigen presenting cells (APCs) from porcine Peyer's patches (PP).Objective: In view of the critical importance of PIE-APCs interactions in the regulation of intestinal immune responses, we aimed to examine the effect of LjTL2937 on activation patterns of APCs from swine PPs in co-cultures with PIE cells. In addition, we investigated whether LjTL2937 was able to beneficially modulate intestinal immunity of piglets after weaning to improve immune-health status.Results: Stimulation of PIE-APCs co-cultures with LjTL2937 increased the expression of MHC-II, CD80/86, IL-10, and Bcl-3 in CD172a+CD11R1- and CD172a+CD11R1high APCs. In addition, the TL2937 strain caused the upregulation of three negative regulators of TLR4 in PIE cells: MKP-1, Bcl-3 and A20. These changes significantly reduced the inflammatory response triggered by TLR4 activation in PIE-APCs co-cultures. The in vivo experiments using castrated male piglets (crossbreeding (LWD) with Landrace (L), Large Yorkshire (W) and Duroc (D))of 3 weeks of age demonstrated that feeding with LjTL2937 significantly reduced blood complement activity and C reactive protein concentrations while no changes were observed in blood leukocytes, ratio of granulocytes to lymphocyte numbers, macrophages' activity and antibody levels. In addition, treatment with LjTL2937 significantly improved growth performance and productivity, and increased carcass quality.Conclusions: We demonstrated that the use of immunobiotics strains like LjTL2937, as supplemental additives for piglets feedings, could be used as a strategy to maintain and improve intestinal homeostasis; that is important for the development of the pig and for health and performance throughout the productive life of the animal.Fil: Suda, Yoshihito. Miyagi University. Department of Food, Agriculture and Environment; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Takahashi, Yu. Miyagi University. Department of Food, Agriculture and Environment; JapónFil: Hosoya, Shoichi. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; JapónFil: Tomosada, Yohsuke. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; JapónFil: Tsukida, Kohichiro. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; JapónFil: Shimazu, Tomoyuki. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Breading and Genetics ; JapónFil: Aso, Hisashi. Tohoku University. Graduate School of Agricultural Science. Cell Biology Laboratory; JapónFil: Tohno, Masanori. National Institute of Livestock and Grassland Science; JapónFil: Ishida, Mitsuharu. Miyagi University. Department of Food, Agriculture and Environment; JapónFil: Makino, Seiya. No especifíca;Fil: Ikegami, Shuji. No especifíca;Fil: Kitazawa, Haruki. Tohoku University. Graduate School of Agricultural Science. Laboratory of Animal Products Chemistry. Food and Feed Immunology Group; Japó

Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells

This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a+CD11R1high and CD4+ cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response

Advanced application of bovine intestinal epithelial cell line for evaluating regulatory effect of lactobacilli against heat-killed enterotoxigenicEscherichia coli-mediated inflammation

Background: Previously, a bovine intestinal epithelial cell line (BIE cells) was successfully established. This work hypothesized that BIE cells are useful in vitro model system for the study of interactions of microbial- or pathogenassociated molecular patterns (MAMPs or PAMPs) with bovine intestinal epithelial cells and for the selection of immunoregulatory lactic acid bacteria (LAB). Results: All toll-like receptor (TLR) genes were expressed in BIE cells, being TLR4 one of the most strongly expressed. We demonstrated that heat-stable PAMPs of enterotoxigenic Escherichia coli (ETEC) significantly enhanced the production of IL-6, IL-8, IL-1! and MCP-1 in BIE cells by activating both NF-"B and MAPK pathways. We evaluated the capacity of several lactobacilli strains to modulate heat-stable ETEC PAMPs-mediated inflammatory response in BIE cells. Among these strains evaluated, Lactobacillus casei OLL2768 attenuated heat-stable ETEC PAMPs-induced pro-inflammatory response by inhibiting NF-"B and p38 signaling pathways in BIE cells. Moreover, L. casei OLL2768 negatively regulated TLR4 signaling in BIE cells by up-regulating Toll interacting protein (Tollip) and B-cell lymphoma 3-encoded protein (Bcl-3). Conclusions: BIE cells are suitable for the selection of immunoregulatory LAB and for studying the mechanisms involved in the protective activity of immunobiotics against pathogen-induced inflammatory damage. In addition, we showed that L. casei OLL2768 functionally modulate the bovine intestinal epithelium by attenuating heat-stable ETEC PAMPs-induced inflammation. Therefore L. casei OLL2768 is a good candidate for in vivo studying the protective effect of LAB against intestinal inflammatory damage induced by ETEC infection or heat-stable ETEC PAMPs challenge in the bovine host.Fil: Takanashi, Naoya. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;Fil: Tomosada, Yohsuke. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Tucuman. Centro de Referencia Para Lactobacilos (i); Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;Fil: Murata, Kozue. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;Fil: Takahashi, Takuya. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;Fil: Chiba, Eriko. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;Fil: Tohno, Masanori. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan; National Agriculture and Food Research Organization. National Institute of Livestock and Grassland Science; Japan.;Fil: Tomoyuki Shimazu. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan; Laboratory of Animal Breading and Genetics. Graduate School of Agricultural Science; Japan.;Fil: Aso, Hisashi. Cell Biology Laboratory, Graduate School of Agricultural Science. Tohoku University; Japan.;Fil: Suda, Yoshihito. Department of Food, Agriculture and Environment. Miyagi University; Japan.;Fil: Ikegami, Shuji. Division of Research and Development. Food Science Institut. Meiji Dairies CoOdawara; Japan;Fil: Itoh, Hiroyuki. Division of Research and Development. Food Science Institut. Meiji Dairies CoOdawara; Japan;Fil: Kawai, Yasushi. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;Fil: Tadao Saito. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan;Fil: Alvarez, Gladis Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina;Fil: Kitazawa, Haruki. Food and Feed Immunology Group. Laboratory of Animal Products Chemistry. Graduate School of Agricultural Science. Tohoku University; Japan

Paraimmunobiotic bifidobacteria modulate the expression patterns of peptidoglycan recognition proteins in porcine intestinal epitheliocytes and antigen presenting ells

Peptidoglycan recognition proteins (PGLYRPs) are a family of pattern recognition receptors (PRRs) that are able to induce innate immune responses through their binding to peptidoglycan (PGN), lipopolysaccharide, or lipoteichoic acid, or by interacting with other PRR-ligands. Recently, progress has been made in understanding the immunobiology of PGLYRPs in human and mice, however, their functions in livestock animals have been less explored. In this study, we characterized the expression patterns of PGLYRPs in porcine intestinal epithelial (PIE) cells and antigen-presenting cells (APCs) and their modulation by the interactions of host cells with PRR-ligands and non-viable immunomodulatory probiotics referred to as paraimmunobiotics. We demonstrated that PGLYRP-1, -2, -3, and -4 are expressed in PIE cells and APCs from Peyer?s patches, being PGLYPR-3 and -4 levels higher than PGLYRP-1 and -2. We also showed that PGLYRPs expression in APCs and PIE cells can be modulated by different PRR agonists. By using knockdown PIE cells for TLR2, TLR4, NOD1, and NOD2, or the four PGLYRPs, we demonstrated that PGLYRPs expressions would be required for activation and functioning of TLR2, TLR4, NOD1, and NOD2 in porcine epitheliocytes, but PGLYRPs activation would be independent of those PRR expressions. Importantly, we reported for the first time that PGLYRPs expression can be differentially modulated by paraimmunobiotic bifidobacteria in a strain-dependent manner. These results provide evidence for the use of paraimmunobiotic bifidobacteria as an alternative for the improvement of resistance to intestinal infections or as therapeutic tools for the reduction of the severity of inflammatory damage in diseases in which a role of PGLYRPs-microbe interaction has been demonstrated.Fil: Iida, Hikaru. Tohoku University; JapónFil: Tohno, Masanori. National Agriculture and Food Research Organization. Central Region Agricultural Research Centre; JapónFil: Islam, Md. Aminul. Tohoku University; Japón. Agricultural University. Faculty of Veterinary Science. Department of Medicine; BangladeshFil: Sato, Nana. Tohoku University; JapónFil: Kobayashi, Hisakazu. Tohoku University; JapónFil: Albarracín, Leonardo Miguel. Tohoku University; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Humayun Kober, AKM. Tohoku University; JapónFil: Ikeda-Ohtsubo, Wakako. Tohoku University; JapónFil: Suda, Yoshihito. Miyagi University. Department of Food, Agriculture and Environment; JapónFil: Aso, Hisashi. Tohoku University; JapónFil: Nochi, Tomonori. Tohoku University; JapónFil: Miyazaki, Ayako. National Institute of Animal Health. Viral Diseases and Epidemiology Research Division; JapónFil: Uenishi, Hirohide. National Agriculture And Food Research Organization; JapónFil: Iwabuchi, Noriyuki. Morinaga Milk Industry Co., Ltd.; JapónFil: Xiao, Jin-zhong. Morinaga Milk Industry Co., Ltd.; JapónFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; JapónFil: Kitazawa, Haruki. Tohoku University; Japó