Yutaka Makimura

Natural killer cell activities of synbiotic Lactobacillus casei ssp. casei in conjunction with dextran

We have reported previously that Lactobacillus casei ssp. casei, together with specific substrate dextran, exhibited an adjuvant effect of stimulating humoral immune responses against bovine serum albumin (BSA) as a model antigen in BALB/c mice. In the present study, among the Lactobacillus species tested, L. casei ssp. casei with dextran significantly elevated the natural killer (NK) cell activites in spleen mononuclear cells from BALB/c mice in comparison to L. casei ssp. casei alone or other Lactobacillus species with or without dextran. Oral administration of L. casei ssp. casei together with dextran also resulted in a significant increase of NK cell activities in healthy human volunteers. Further, L. casei ssp. casei induced significant production of interleukin (IL)‐12 in human peripheral blood mononuclear cells and IL‐15 mRNA expression in the human intestinal epithelial cell line Caco‐2. L. casei ssp. casei with dextran in food also significantly elevated the survival rate of BALB/c mice bearing Meth‐A cells. Taken together, these results demonstrate that dietary synbiotic supplementation which is a combination of the L. casei ssp. casei used as a probiotic together with the dextran, a specific substrate as a prebiotic, efficiently elicits murine and human NK cell activities.

Toll‐like receptor 4‐dependent recognition of structurally different forms of chemically synthesized lipid As of Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram‐negative anaerobic oral black‐pigmented bacterium closely associated with chronic periodontitis. Lipopolysaccharide (LPS) derived from P. gingivalis is shown to be unusual because the LPS contains a greater number of lipid A species, such as tri‐, tetra‐, and/or penta‐acylated lipid As. In this study, a lipid A possessing penta‐fatty acyl chains derived from P. gingivalis strain 381 (compound PG‐381–5FA) was synthesized, and examined for its immunobiological activities, compared with a tri‐acylated lipid A (compound PG‐381–3FA) synthesized previously. Compound PG‐381‐5FA, similar to compound PG‐381‐3FA, demonstrated weaker activity in a Limulus test as compared with Escherichia coli‐type synthetic lipid A (compound 506). Compound PG‐381‐5FA, followed by compound PG‐381‐3FA, induced KC, interleukin‐6, and tumour necrosis factor‐α production in peritoneal macrophages from LPS‐responsive C3H/HeN mice, but not in those from LPS‐hyporesponsive C3H/HeJ mice. Furthermore, compound PG‐381‐5FA, as well as compound PG‐381‐3FA, activated nuclear factor‐κB via Toll‐like receptor (TLR)4/mD‐2, but not TLR2, in a manner similar to compound 506, and worked as an antagonist for compound 506‐induced cell activation. In the case of human peripheral blood mononuclear cells, compound PG‐381‐5FA showed much stronger IL‐6‐inducing activity than compound PG‐381‐3FA. The present results demonstrate that the chemical synthesis of a penta‐acylated lipid A, mimicking the natural lipid A portion of LPS from P. gingivalis, is attributable to immune cell activation through TLR4, similar to that of compound 506.

Soluble CD14 Discriminates Slight Structural Differences between Lipid As That Lead to Distinct Host Cell Activation

Soluble CD14 (sCD14) in serum is known to sensitize host cells to LPS. In the present study, the contributions of sCD14 and LPS-binding protein to a lipid A moiety from LPS preparations of periodontopathogenic Fusobacterium nucleatum sp. nucleatum were compared with that of Escherichia coli-type synthetic lipid A (compound 506). F. nucleatum lipid A was identified to be a hexa-acylated fatty acid composed of tetradecanoate (C14) and hexadecanoate (C16), similar to dodecanoate (C12) and C14 in compound 506. The two lipid A specimens exhibited nearly the same reactivity in Limulus amoebocyte lysate assays, though F. nucleatum lipid A showed a weaker lethal toxicity. Both lipid A specimens showed nearly the same activities toward host cells in the absence of FBS, though compound 506 exhibited much stronger activity in the presence of FBS, sCD14, or sCD14 together with LPS-binding protein. Furthermore, native PAGE/Western immunoblot assays demonstrated that F. nucleatum lipid A had a weaker binding to sCD14 as compared with compound 506. These results suggest that sCD14 is able to discriminate the slight structural differences between these lipid As, which causes their distinct host cell activation activities.

Correlation between chemical structure and biological activities of Porphyromonas gingivalis synthetic lipopeptide derivatives.

We recently separated a PG1828‐encoded triacylated lipoprotein (Pg‐LP), composed of two palmitoyl and one pentadecanoyl groups at the N‐terminal of glycerocysteine from Porphyromonas gingivalis, a periodontopathic bacteria, and found that Pg‐LP exhibited definite biological activities through Toll‐like receptor (TLR) 2. In the present study, we synthesized 12 different Pg‐LP N‐terminal peptide moieties (PGTP) using four combinations of glyceryl (R and S) and cysteinyl (l and d) stereoisomers, and three different acyl group regioisomers, N‐pentadecanoyl derivative (PGTP1), S‐glycero 2‐pentadecanoyl derivative (PGTP2) and S‐glycero 3‐pentadecanoyl derivative (PGTP3). All the PGTP compounds (RL, SL, SD, RD) tested showed TLR2‐dependent cell activation. The activating capacities of the PGTP‐R compounds were more potent than those of the PGTP‐S compounds, whereas there were no differences between the PGTP‐L and ‐D compounds. Furthermore, the production of interleukin (IL)‐6 following stimulation with the PGTP1‐RL, PGTP2‐RL and PGTP3‐RL compounds was impaired in peritoneal macrophages from TLR2 knock‐out (KO), but not those from TLR1 KO or TLR6 KO mice. These results suggest that P. gingivalis triacylated lipopeptides are capable of activating host cells in a TLR2‐dependent and TLR1‐/TLR6‐independent manner, and the fatty acid residue at the glycerol position in the PGTP molecule plays an important role in recognition by TLR2.

Treponema medium Glycoconjugate Inhibits Activation of Human Gingival Fibroblasts Stimulated with Phenol-Water Extracts of Periodontopathic Bacteria:

Oral treponemes are well-known as causative agents of periodontal diseases; however, the details have not been fully clarified. Here, we examined the effects of Treponema medium glycoconjugate on the activation of human gingival fibroblasts using phenol-water extracts from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum subsp. nucleatum, and Actinobacillus actinomycetemcomitans. The phenol-water extracts activated human gingival fibroblasts to mediate IL-8 production, as well as IL-8 mRNA expression, phosphorylation of p38 mitogen-activated protein kinase, and expression of intercellular adhesion molecule-1. T. medium glycoconjugate exhibited no activation of human gingival fibroblasts, while phenol-water extract-induced activation of human gingival fibroblasts was clearly inhibited by T. medium glycoconjugate. Furthermore, binding of biotinylated phenol-water extracts to CD14 in the presence of LPS-binding protein was blocked with T. medium glycoconjugate. These results suggest that T. medium glycoconjugate has an inhibitory effect on host cell activation by periodontopathic bacteria caused by binding to CD14- and LPS-binding protein.