Takeshi Shimosato

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.

Immunobiotic Lactobacillus jensenii Elicits anti-inflammatory activity in porcine intestinal epithelial cells by modulating negative regulators of the toll-like receptor signaling pathway

ABSTRACT The effect of Lactobacillus jensenii TL2937 on the inflammatory immune response triggered by enterotoxigenic Escherichia coli (ETEC) and lipopolysaccharide (LPS) in a porcine intestinal epitheliocyte cell line (PIE cells) was evaluated. Challenges with ETEC or LPS elicited Toll-like receptor 4 (TLR4)-mediated inflammatory responses in cultured PIE cells, indicating that our cell line may be useful for studying inflammation in the guts of weaning piglets. In addition, we demonstrated that L. jensenii TL2937 attenuated the expression of proinflammatory cytokines and chemokines caused by ETEC or LPS challenge by downregulating TLR4-dependent nuclear factorκB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. Furthermore, we demonstrated that L. jensenii TL2937 stimulation of PIE cells upregulated three negative regulators of TLRs: A20, Bcl-3, and MKP-1, deepening the understanding of an immunobiotic mechanism of action. L. jensenii TL2937-mediated induction of negative regulators of TLRs would have a substantial physiological impact on homeostasis in PIE cells, because excessive TLR inflammatory signaling would be downregulated. These results indicated that PIE cells can be used to study the mechanisms involved in the protective activity of immunobiotics against intestinal inflammatory damage and may provide useful information for the development of new immunologically functional feeds that help to prevent inflammatory intestinal disorders, including weaning-associated intestinal inflammation.

Accelerated wound healing mediated by activation of Toll‐like receptor 9

Wound healing is mediated through complex interactions between circulating immune cells and local epithelial and endothelial cells. Elements of the innate immune system are triggered when Toll‐like receptors (TLR) are stimulated by their cognate ligands, and previous studies suggest that such interactions can accelerate wound healing. This work examines the effect of treating excisional skin biopsies with immunostimulatory CpG oligodeoxynucleotides (ODN) that trigger via TLR9. Results indicate that CpG (but not control) ODN accelerate wound closure and reduce the total wound area exposed over time by >40% (p<0.01). TLR9 knockout mice, a strain unresponsive to the immunomodulatory effects of CpG stimulation, are unresponsive to ODN treatment and exhibit a general delay in healing when compared with wild‐type mice. CpG ODN administration promoted the influx of macrophages to the wound site and increased the production of vascular endothelial growth factor, expediting neovascularization of the wound bed (p<0.01 for both parameters). Stimulation via TLR9 thus represents a novel strategy to accelerate wound healing.

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.

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. 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.

Effect of Probiotics/Prebiotics on Cattle Health and Productivity

Probiotics/prebiotics have the ability to modulate the balance and activities of the gastrointestinal (GI) microbiota, and are, thus, considered beneficial to the host animal and have been used as functional foods. Numerous factors, such as dietary and management constraints, have been shown to markedly affect the structure and activities of gut microbial communities in livestock animals. Previous studies reported the potential of probiotics and prebiotics in animal nutrition; however, their efficacies often vary and are inconsistent, possibly, in part, because the dynamics of the GI community have not been taken into consideration. Under stressed conditions, direct-fed microbials may be used to reduce the risk or severity of scours caused by disruption of the normal intestinal environment. The observable benefits of prebiotics may also be minimal in generally healthy calves, in which the microbial community is relatively stable. However, probiotic yeast strains have been administered with the aim of improving rumen fermentation efficiency by modulating microbial fermentation pathways. This review mainly focused on the benefits of probiotics/prebiotics on the GI microbial ecosystem in ruminants, which is deeply involved in nutrition and health for the animal.

Suppressive Oligodeoxynucleotides Inhibit Silica-Induced Pulmonary Inflammation

Inhalation of silica-containing dust particles induces silicosis, an inflammatory disease of the lungs characterized by the infiltration of macrophages and neutrophils into the lungs and the production of proinflammatory cytokines, chemokines, and reactive oxygen species (ROS). Synthetic oligodeoxynucleotides (ODN) expressing “immunosuppressive motifs” were recently shown to block pathologic inflammatory reactions in murine models of autoimmune disease. Based on those findings, the potential of suppressive ODN to prevent acute murine silicosis was examined. In vitro studies indicate that suppressive ODN blunt silica-induced macrophage toxicity. This effect was associated with a reduction in ROS production and p47phox expression (a subunit of NADPH oxidase key to ROS generation). In vivo studies show that pretreatment with suppressive (but not control) ODN reduces silica-dependent pulmonary inflammation, as manifest by fewer infiltrating cells, less cytokine/chemokine production, and lower levels of ROS (p < 0.01 for all parameters). Treatment with suppressive ODN also reduced disease severity and improved the survival (p < 0.05) of mice exposed to silica.

Dextran from Leuconostoc mesenteroides Augments Immunostimulatory Effects by the Introduction of Phosphate Groups

The immunological effects of phosphorylated dextran (in which phosphate groups were chemically introduced) on murine splenocytes were examined. When dextran produced by Leuconostoc mesenteroides was phosphorylated by a reaction with polyphosphoric acid in formamide solution for 48 h, the degree of phosphorylation of dextran was maximal. The highest phosphorus content (1.7%, wt/wt) was observed in 40 kDa of dextran. The mitogenic response of murine splenocytes was enhanced by the phosphorylated dextran, but its activity was not related to its molecular weight. A strong response was detected at a concentration of 10 to 500 microg/ml, and the highest activity was obtained 48 h after stimulation. Phosphorylated dextran was characterized as a B-cell-specific mitogen. The expressions of CD86 on CD8alpha- CD11c- and CD8alpha- CD11c+ cells were augmented by phosphorylated dextran. The levels of mRNA expression of gamma interferon and interleukin-10 on murine splenocytes were also increased by the stimulation. These results demonstrate that dextran exerts immunostimulation by the introduction of phosphate groups.

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 Peyers 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.