Masanori Tohno

Immunostimulatory Oligodeoxynucleotide Containing TTTCGTTT Motif from Lactobacillus rhamnosus GG DNA Potentially Suppresses OVA‐specific IgE Production in Mice

In a previous study of the immunoregulatory properties of commensal bacterial DNA, we identified the strong immunostimulatory oligodeoxynucleotide (ISS‐ODN) ID35 in the genomic DNA of Lactobacillus rhamnosus GG (LGG). The observed effects of ID35 are because of the unique TTTCGTTT motif located at the 5′ end of the ODN, which is different from the previously identified ISS motifs in humans and mice. In the present study, we used an ovalbumin (OVA)‐sensitized mouse model to show that ID35 is a potent suppressor of antigen‐specific immunoglobulin E (IgE) production in vivo. This effect was toll‐like receptor 9‐dependent, as GpC negID35 failed to suppress antigen‐specific IgE production. ID35 activated the specific subset of CD11c+CD8a+ dendritic cells, which are associated with T‐helper 1 (Th1)‐type systemic responses, and effectively induced interferon‐gamma (IFN‐γ) production by CD4+ T cells in OVA‐challenged mice. These immunoregulatory effects of ID35 were comparable with those induced by the murine prototype ODN 1826. Thus, ID35 is the first ISS‐ODN with such a strong immunostimulatory and IgE suppressor activity to be found in immunobiotic bacterial DNA.

Strong immunostimulatory activity of AT-oligodeoxynucleotide requires a six-base loop with a self-stabilized 5′-C…G-3′ stem structure

Lactobacillus gasseri OLL2716 has recently been discovered as a probiotic that suppresses the growth of Helicobacter pylori and reduces gastric mucosal inflammation in humans. This has resulted in the development of a new type of probiotic yoghurt ‘LG21’ in Japan. In our previous study, we found an immunostimulatory AT5ACL oligodeoxynucleotide (AT‐ODN) containing a unique core sequence (5′‐ATTTTTAC‐3′) in L.u2003gasseri JCM1131T. Interestingly, although the AT‐ODN does not contain any CpG sequences, it exerts mitogenic activity in B cells and augments Th‐1‐type immune responses via Toll‐like receptor 9. These findings prompted us to identify strong immunostimulatory non‐CpG AT‐ODNs that contain the 5′‐ATTTTTAC‐3′ motif in the genomic sequence of L.u2003gasseri OLL2716. We identified 280 kinds of AT‐ODNs in the L.u2003gasseri OLL2716 genome. Mitogenicity and NF‐κB gene reporting assays showed that 13 of the 280 AT‐ODNs were strongly immunostimulatory when in the TLR9 transfectant. Of these, AT‐ODNs LGAT‐145 and LGAT‐243 were the most potent. With respect to the induction of Th‐1‐type cytokines, LGAT‐243 had the greatest activity and was more potent than the swine prototype, ODN D25. We further found that a six‐base secondary loop structure containing a self‐stabilized 5′‐C…G‐3′ stem sequence is important for potent immunostimulatory activity. These results show for the first time that AT‐ODNs with a specific loop and stem structure are important factors for immunostimulatory activity. Finally, we found that novel strong immunostimulatory non‐CpG AT‐ODNs exist in the genome of probiotic lactic acid bacteria.

Immunomodulation in gut-associated lymphoid tissue of neonatal chicks by immunobiotic diets

Developmental changes in immunocompetent cells of the gut during the first week posthatch were determined in broiler chicks fed immunobiotic lactic acid bacteria in the form of Lactobacillus jensenii TL2937-, Lactobacillus gasseri JCM1131(T)-, Lactobacillus delbrueckii ssp. bulgaricus NIAIB6-, or L. gasseri TL2919-supplemented diets. The relative weights of spleen and bursa of Fabricius in chicks fed the immunobiotic diets were slightly higher than the control valued at 1 and 3 d of age, with the exception of spleen weight in the L. gasseri JCM1131(T) at 3 d of age, the bursa of Fabricius weight in the L. gasseri JCM1131(T) at 1 and 3 d of age, and bursa of Fabricius weight in the L. gasseri TL2919 group at 1 d of age. There were no significant differences in body and liver weights among the treatments. When chicks were fed the L. jensenii TL2937- or L. gasseri TL2919-supplemented diets, expression of T cell-related mRNA [cluster of differentiation 3 (CD3), interleukin-2 (IL-2), and interferon-gamma (IFN-gamma)] in the foregut was significantly higher than that of control chicks at 3 or 7 d of age. Expression levels of toll-like receptor (TLR) mRNA tended to increase in the foregut of chicks fed the immunobiotic diets, except for the L. delbrueckii ssp. bulgaricus NIAIB6, compared with expression levels in control chicks. The Bu-1 mRNA expression levels in the bursa of Fabricius were not affected by the supplementations with immunobiotic lactic acid bacteria. These results show that immunobiotics, particularly L. gasseri TL2919, might be useful as immunomodulators to stimulate the gut-associated immune system in neonatal chicks, and thereby protect them from disease without decreasing growth performance as a possible substitution of antibiotics.

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

BackgroundPreviously, 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 pathogen-associated molecular patterns (MAMPs or PAMPs) with bovine intestinal epithelial cells and for the selection of immunoregulatory lactic acid bacteria (LAB).ResultsAll 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-1α and MCP-1 in BIE cells by activating both NF-κB 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-κB 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).ConclusionsBIE 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.

Utilization of the Porcine System to Study Lymphotoxin-β Regulation in Intestinal Lymphoid Tissue

Lymphotoxin-β (LT-β) has been suggested to be a regulator of secondary lymphoid structure development. In the present study, we isolated porcine LT-β (poLT-β) from adult swine spleens. The open reading frame encoded a predicted 246-amino acid polypeptide exhibiting higher similarity to the human than the mouse LT-β protein. Expression of LT-β mRNA in various swine tissues was analyzed by real-time PCR, and it was found to be higher in the ileal Peyers patches (Pps) of adults than in newborns. In addition, ligand stimulation of toll-like receptors 2, 4, and 9, which are activated by bacterial components, increased LT-β expression only in neonatal ileal Pps. These results suggest that colonization by commensal bacteria may affect the maturation of neonatal ileal Pps by the induction of LT-β via toll-like receptors. LT-β may therefore be useful for studying the development of the intestinal immune system at parturition in both swine and humans.