Shinichiro Katoh

Strong immunostimulation in murine immune cells by Lactobacillus rhamnosus GG DNA containing novel oligodeoxynucleotide pattern.

Whole cells, cell wall components and some soluble factors from Lactobacillus rhamnosus GG (LGG) are known to invoke immune responses as they interact with animal and human immune cells. In the present study, we found that chromosomal DNA from LGG is a potent inducer of splenic B cell proliferation, CD86/CD69 expression and cytokine production in mice. In the genomic DNA of LGG we discovered TTTCGTTT oligodeoxynucleotide (ODN) ID35, which has a potent activity in a number of immunostimulatory assays. Phosphorothioate backbone is not required for the activity of ID35. The ODN ID35 showed levels of activity comparable with those induced by the murine prototype ODN 1826 in B cell proliferation, CD86/CD69 expression, interleukin (IL)‐6, IL‐12, IL‐18, interferon gamma (IFN‐γ) and tumour necrosis factor alpha (TNF‐α) mRNA expression and IFN‐γ/IL‐12p70 protein production assays. Additionally, ID35 appeared to be equally active in both murine and human immune cells. These stimulatory effects are due to TTTCGTTT motif located in the 5′ end of ID35. In this study we demonstrate for a first time that, DNA from LGG is a factor of immunobiotic activity. Furthermore, ODN ID35 is the first ODN, with such a strong immunostimulatory activity to be found in immunobiotic bacterial DNA.

Swine Toll-like receptor 9 recognizes CpG motifs of human cell stimulant

Complementary DNA (cDNA) encoding swine Toll-like receptor 9 (sTLR9) was isolated from Peyers patches (Pps) of gut-associated lymphoid tissue (GALT). The complete open reading frame (ORF) of sTLR9 contains 3093 bp coding deduced 1030 amino acid residues. The amino acid sequence of sTLR9 was characterized by a signal peptide followed by multiple leucine-rich repeats, a transmembrane sequence and a cytoplasmic domain homologous to that of the human interleukin-1 receptor (TIR). The sTLR9 showed a higher amino acid identity with humans (81.8%) and felis catus (86.7%) than mice (74.9%). The HEK293T cells transfected with pCXN2.1-FLAG DNA containing the sTLR9 cDNA were expressed sTLR9 as a membrane-bound molecules, which were reactive with anti-sTLR9 rabbit polyclonal antibody. Moreover, the transfectant was responsible for the CpG oligo DNA. sTLR9 was preferentially expressed in Pps and mesenteric lymph nodes (MLNs), and its degree was approximately three times higher than a spleen but weak in the other tissues by the real-time quantitative PCR analyses. The strong expression of sTLR9 in Pps and MLNs and its recognizing CpG DNA for human cell stimulant are shown first in this study, which may help in understanding the intestinal immune system mediated by a bacterial DNA through TLR9.

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. gasseri 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. gasseri OLL2716. We identified 280 kinds of AT‐ODNs in the L. gasseri 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.

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.