Yoshinari Yamamoto

Generation of Dipeptidyl Peptidase-IV-Inhibiting Peptides from β-Lactoglobulin Secreted by Lactococcus lactis

Previous studies showed that hydrolysates of β-lactoglobulin (BLG) prepared using gastrointestinal proteases strongly inhibit dipeptidyl peptidase-IV (DPP-IV) activity in vitro. In this study, we developed a BLG-secreting Lactococcus lactis strain as a delivery vehicle and in situ expression system. Interestingly, trypsin-digested recombinant BLG from L. lactis inhibited DPP-IV activity, suggesting that BLG-secreting L. lactis may be useful in the treatment of type 2 diabetes mellitus.

Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis

Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.

Development of a simple IgE-independent anaphylactic model using buckwheat antigen and B-type CpG oligodeoxynucleotide from Streptococcus thermophilus.

We developed a severe anaphylactic model in mice using buckwheat antigen and B-type CpG-oligodeoxynucleotides (CpG-ODNs) from Streptococcus thermophilus genome. In typical systemic anaphylaxis models, animals are challenged with large quantity of antigens via an intravenous (i.v.) route. Here, we showed a simple anaphylactic shock after challenge via intraperitoneal (i.p.) route. The i.p. method is simpler than i.v. administration and has a lower risk for failure. To generate this anaphylactic model, 5-week-old female BALB/c mice were first i.p. sensitized with buckwheat antigen mixed with B-type CpG-ODN. After 2 weeks, mice were challenged with antigen to induce anaphylactic shock, which was evaluated by scoring the severity symptoms and measuring serum levels of various proteins and splenic cell producing cytokines. Immunoglobulin (Ig)G2a production and interferon-γ positive cells were markedly increased in mice immunized with antigen mixed with B-type CpG-ODN, whereas serum IgE levels were decreased by B-type CpG-ODN. We also examined the effects of various ODNs (A, B and C-type CpG-ODNs) and antigens (buckwheat, α-casein, β-lactoglobulin and ovalbumin) on anaphylactic severity, and found that the combination of buckwheat and B-type CpG-ODN induced the most intense anaphylactic shock. This model is expected to contribute to the study of the prevention of anaphylactic shock.

Genetically modified Lactococcus lactis producing a green fluorescent protein–bovine lactoferrin fusion protein suppresses proinflammatory cytokine expression in lipopolysaccharide-stimulated RAW 264.7 cells

Lactoferrin (LF), an iron-binding glycoprotein distributed widely in the biological fluids of mammals, is believed to play an important role in host defenses against infection. Previous studies in animal models and humans demonstrated that combined administration of LF and probiotic lactic acid bacteria (LAB) can prevent sepsis. In this study, we genetically engineered a probiotic LAB strain, Lactococcus lactis, to produce recombinant bovine LF based on the green fluorescent protein (GFP)-fused expression system. Western blotting confirmed that the genetically modified L. lactis strain (designated NZ-GFP-bLF) produced a protein corresponding to a fusion of GFP and bLF in the presence of nisin, an inducer of target gene expression. The protein synthesized by NZ-GFP-bLF was fluorescent and thus we monitored the time-dependent change in the production level of the recombinant protein using fluorometric analysis. The utility of NZ-GFP-bLF in preventing sepsis was determined by investigating its anti-inflammatory property in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. Pretreatment of RAW 264.7 cells with NZ-GFP-bLF significantly attenuated the LPS-induced mRNA expression and protein production of 3 proinflammatory cytokines (IL-1α, IL-6, and tumor necrosis factor-α) compared with pretreatment with a vector control strain of L. lactis. Our results suggest that NZ-GFP-bLF holds promise for the development of a new prophylaxis for sepsis.

Class A CpG Oligonucleotide Priming Rescues Mice from Septic Shock via Activation of Platelet-Activating Factor Acetylhydrolase

Sepsis is a life-threatening, overwhelming immune response to infection with high morbidity and mortality. Inflammatory response and blood clotting are caused by sepsis, which induces serious organ damage and death from shock. As a mechanism of pathogenesis, platelet-activating factor (PAF) induces excessive inflammatory responses and blood clotting. In this study, we demonstrate that a Class A CpG oligodeoxynucleotide (CpG-A1585) strongly induced PAF acetylhydrolase, which generates lyso-PAF. CpG-A1585 rescued mice from acute lethal shock and decreased fibrin deposition, a hallmark of PAF-induced disseminated intravascular coagulation. Furthermore, CpG-A1585 improved endotoxin shock induced by lipopolysaccharide, which comprises the cell wall of Gram-negative bacteria and inhibits inflammatory responses induced by cytokines such as interleukin-6 and tumor necrosis factor-α. These results suggest that CpG-A1585 is a potential therapeutic target to prevent sepsis-related induction of PAF.

Recombinant Mouse Osteocalcin Secreted by Lactococcus lactis Promotes Glucagon-Like Peptide-1 Induction in STC-1 Cells

An osteoblastic protein, osteocalcin (OC), exists in vivo in two forms: carboxylated OC, and uncarboxylated or low-carboxylated OC (ucOC). ucOC acts as a hormone to regulate carbon and energy metabolism. Recent studies demonstrated that ucOC exerts insulinotropic effects, mainly through the glucagon-like peptide 1 (GLP-1) pathway. GLP-1 is an insulinotropic hormone secreted by enteroendocrine L cells in the small intestine. Thus, efficient delivery of ucOC to the small intestine may be a new therapeutic option for metabolic diseases such as diabetes and obesity. Here, we genetically engineered a lactic acid bacterium, Lactococcus lactis, to produce recombinant mouse ucOC. Western blotting showed that the engineered strain (designated NZ-OC) produces and secretes the designed peptide (rOC) in the presence of nisin, an inducer of the recombinant gene. Highly-purified rOC was obtained from the culture supernatants of NZ-OC using immobilized metal affinity chromatography. An in vitro assay showed that purified rOC promotes GLP-1 secretion in a mouse intestinal neuroendocrine cell line, STC-1, in a dose-dependent manner. These results clearly demonstrate that NZ-OC secretes rOC, and that rOC can promote GLP-1 secretion by STC-1 cells. Genetically modified lactic acid bacteria (gmLAB) have been proposed over the last two decades as an effective and low-cost mucosal delivery vehicle for biomedical proteins. NZ-OC may be an attractive tool for the delivery of rOC to trigger GLP-1 secretion in the small intestine to treat diabetes and obesity.

Synergistic oligodeoxynucleotide strongly promotes CpG-induced interleukin-6 production

BackgroundBacterial genomes span a significant portion of diversity, reflecting their adaptation strategies; these strategies include nucleotide usage biases that affect chromosome configuration. Here, we explore an immuno-synergistic oligodeoxynucleotide (iSN-ODN, named iSN34), derived from Lactobacillus rhamnosus GG (LGG) genomic sequences, that exhibits a synergistic effect on immune response to CpG-induced immune activation.MethodsThe sequence of iSN34 was designed based on the genomic sequences of LGG. Pathogen-free mice were purchased from Japan SLC and maintained under temperature- and light-controlled conditions. We tested the effects of iSN34 exposure in vitro and in vivo by assessing effects on mRNA expression, protein levels, and cell type in murine splenocytes.ResultsWe demonstrate that iSN34 has a significant stimulatory effect when administered in combination with CpG ODN, yielding enhanced interleukin (IL)-6 expression and production. IL-6 is a pleotropic cytokine that has been shown to prevent epithelial apoptosis during prolonged inflammation.ConclusionsOur results are the first report of a bacterial-DNA-derived ODN that exhibits immune synergistic activity. The potent over-expression of IL-6 in response to treatment with the combination of CpG ODN and iSN34 suggests a new approach to immune therapy. This finding may lead to novel clinical strategies for the prevention or treatment of dysfunctions of the innate and adaptive immune systems.

Recombinant mouse calcitonin gene-related peptide secreted by Lactococcus lactis inhibits lipopolysaccharide-induced inflammatory response in macrophages

We describe the development of a genetically modified strain of lactic acid bacteria (gmLAB) capable of producing a recombinant mouse calcitonin gene-related peptide (rCGRP). This strain (NZ-CGRP) was generated by introducing a CGRP secretion plasmid into Lactococcus lactis NZ9000. Western blotting confirmed the secretion of rCGRP in the presence of the inducer nisin. Highly purified rCGRP was obtained from the culture supernatants of NZ-CGRP. We demonstrated that prophylactic exposure of a culture of mouse peritoneal macrophages to rCGRP inhibited lipopolysaccharide (LPS) induction of tumor necrosis factor-α (TNF-α). The rCGRP-secreting gmLAB strain holds promise for development as a new anti-inflammatory prophylactic.

Immune synergistic oligodeoxynucleotide from Lactobacillus rhamnosus GG enhances the immune response upon co-stimulation by bacterial and fungal cell wall components

Bacterial genomic DNA has recently been shown to elicit a highly evolved immune defense. This response can be selectively triggered for a wide range of therapeutic applications, including use as a vaccine adjuvant to immunotherapies for allergy, cancer, and infectious diseases. Previously, we identified a low-concentration immune synergistic oligodeoxynucleotide (iSN-ODN, named iSN34) from Lactobacillus rhamnosus GG that has immunosynergistic activity upon costimulation of target cells with ligands of Toll-like receptor 9 (TLR9). Here, we extend that observation by demonstrating the synergistic induction (in mouse splenocytes) of IL-6 by the combination of iSN34 with cell wall components of bacteria and fungi. We observed that splenocytes pretreated with iSN34 and then costimulated with agonists for TLR1/2 (Pam3 CSK4 ), TLR4 (lipopolysaccharide), or TLR2/6 (Zymosan) exhibited enhanced accumulation of IL-6. These results suggested that the combination of iSN34 with TLR1/2, TLR4, or TLR2/6 agonists may permit the induction of a potent immune response.