Ribosome-Engineered Lacticaseibacillus rhamnosus Strain GG Exhibits Cell Surface Glyceraldehyde-3-Phosphate Dehydrogenase Accumulation and Enhanced Adhesion to Human Colonic Mucin.

Differences in individual host responses have emerged as an issue regarding the health benefits of probiotics. Here, we applied ribosome engineering (RE) technology, developed in an actinomycete study, to Lacticaseibacillus rhamnosus GG (LGG). RE can effectively enhance microbial potential by using antibiotics to induce spontaneous mutations in the ribosome and/or RNA polymerase. In this study, we identified eight types of streptomycin resistance mutations in the LGG rpsL gene, which encodes ribosomal protein S12. Notably, LGG harboring the K56N mutant (LGG-MTK56N) expressed high levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the cell surface compared with the LGG wild type (LGG-WT). GAPDH plays a key role in colonic mucin adhesion. Indeed, LGG-MTK56N significantly increased type A human colonic mucin adhesion compared to LGG-WT in experiments using the Biacore system. The ability to adhere to the colon is an important property of probiotics; thus, these results suggest that RE is an effective breeding strategy for probiotic lactic acid bacteria.IMPORTANCE We sought to apply ribosome engineering (RE) to probiotic lactic acid bacteria and to verify RE's impact. Here, we showed that one mutant of RE Lacticaseibacillus rhamnosus GG (LGG-MTK56N) bore a GAPDH on the cell surface; the GAPDH was exported via an ABC transporter. Compared to the wild-type parent, LGG-MTK56N adhered more strongly to human colonic mucin and exhibited a distinct cell size and shape. These findings demonstrate that RE in LGG-MTK56N yielded dramatic changes in protein synthesis, protein transport, and cell morphology and affected adherence to human colonic mucin.

Construction of genetically modified Lactococcus lactis that produces bioactive anti-interleukin-4 single-chain fragment variable.

Interleukin 4 (IL-4) is a cytokine that induces T-cell differentiation and the production of antibodies from B cells, and plays a crucial role in the allergic response. Therefore, development of a therapeutic approach against IL-4 signaling is expected to prevent or control Th2-related allergic diseases. IL-4 single-chain fragment variable (scFv), which is a recombinant protein consisting of the Fv region of an IL-4 antibody connected to a flexible peptide linker, is expected to be an inhibitor of IL-4 signaling. In this study, recombinant IL-4 scFv was produced by genetically modified lactic acid bacteria (gmLAB); this system is gaining attention as a type of microbial therapeutics. Recombinant gene expression was confirmed with western blotting, and the IL-4 recognition ability of IL-4 scFv produced by gmLAB was examined with an enzyme-linked immunosorbent assay. The macrophage cell line, Raw264.7, and peritoneal macrophages isolated from C57BL/6 mice were employed for an in vitro IL-4 signaling inhibition assay. IL-4 stimulation increased the mRNA expression of arginase-1, a biomarker of IL-4 signaling in macrophages, but arginase-1 expression was suppressed by IL-4 scFv produced by gmLAB, indicating that IL-4 scFv has IL-4 signaling inhibitory activity. gmLAB that produces bioactive IL-4 scFv that was constructed in this study could be an attractive approach for treating allergic disorders.

Oral administration of Flavonifractor plautii attenuates inflammatory responses in obese adipose tissue.

Adipose tissue inflammation enhances the symptoms of metabolic syndrome. Flavonifractor plautii, a bacterium present in human feces, has been reported to participate in the metabolism of catechin in the gut. The precise function of F. plautii remains unclear. We assessed the immunoregulatory function of F. plautii both in vitro and in vivo. In vitro, we showed that both viable and heat-killed F. plautii attenuated TNF-α transcript accumulation in lipopolysaccharide-stimulated RAW 264.7 cells. For the in vivo experiment, male C57BL/6 were placed on a high-fat diet (HFD) for 11 weeks. During the final two weeks on the HFD, the animals were administered with F. plautii by once-daily oral gavage. The oral administration of F. plautii attenuated the increase in TNF-α transcription otherwise seen in the epididymal adipose tissue of HFD-fed obese mice (HFD + F. plautii). The composition of the microbial population (at the genus level) in the cecal contents of the HFD + F. plautii mice was altered considerably. In particular, the level of Sphingobium was decreased significantly, and that of Lachnospiraceae was increased significantly, in the HFD + F. plautii group. Obesity is closely associated with the development of inflammation in adipose tissue. F. plautii may be involved in inhibition of TNF-α expression in inflammatory environments. Our results demonstrated that F. plautii may be useful for alleviating the inflammatory responses of adipose tissue.

Regulatory role of heme oxygenase-1 in silica-induced lung injury.

BACKGROUND: Silicosis, a progressive inflammatory lung disease attributed mainly to occupational exposure to silica dust, shows loss of lung function even after cessation of exposure. In addition to conventional evaluation methods such as chest X-ray, computed tomography, and spirometry, we identified heme oxygenase (HO)-1, an inducible antioxidant, as a potential biomarker to identify at-risk patients. We found that HO-1 was critical in attenuating the disease progression of silicosis; however, the key signaling pathway has not yet been elucidated. Here, we report the critical pathway after silica exposure, focusing on the role of silica-derived reactive oxygen species (ROS) signaling and its attenuation, which is mediated by HO-1 induction, in vivo and in vitro. METHODS: Normal bronchial epithelial cells and a macrophage cell line, as well as a murine silicosis model generated by intratracheal administration of 2.5 mg of crystalline silica, were used in this study. The pathways activated in response to silica exposure, including the mitogen-activated protein kinase (MAPK) signaling pathway, were examined and compared with or without super-induction of HO-1. RESULTS: The murine silicosis model was first assessed for the evaluation of activated pathways after silica exposure, focusing on ROS-MAPK activation. In the murine model, increased expression of HO-1 in the lungs was observed after silica-instillation. Moreover, silica-medicated activation of extracellular signal-regulated kinase (ERK) in the lungs was attenuated in response to silica-induced HO-1 upregulation. Activation of other MAPKs, such as p38 and c-Jun N-terminal kinase pathways, after silica exposure was not significantly different irrespective of HO-1 induction. Further in vitro studies showed that 1) silica-induced HO-1 was significantly attenuated by inhibiting ERK activation, and 2) carbon monoxide and bilirubin as final byproducts of HO-1 could inhibit ERK activation. Taken together, silica-induced HO-1 upregulation was mediated by ERK activation, and HO-1 further regulates ERK activation via its final byproducts, carbon monoxide and bilirubin. CONCLUSIONS: This is the first study to demonstrate the regulatory role of HO-1 in silicosis. This finding could contribute to the development of a treatment strategy of monitoring HO-1 levels as a marker of therapeutic intervention.

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.

BACKGROUND: Bacterial 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. METHODS: The 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. RESULTS: We 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. CONCLUSIONS: Our 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.

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.

Inhibitory/suppressive oligodeoxynucleotide nanocapsules as simple oral delivery devices for preventing atopic dermatitis in mice.

Here, we report a simple and low-cost oral oligodeoxynucleotide (ODN) delivery system targeted to the gut Peyer's patches (PPs). This system requires only Dulbecco's modified eagle's medium, calcium chloride, ODNs, and basic laboratory equipment. ODN nanocapsules (ODNcaps) were directly delivered to the PPs through oral administration and were taken up by macrophages in the PPs, where they induced an immune response. Long-term continuous oral dosing with inhibitory/suppressive ODNcaps (iODNcaps, "iSG3caps" in this study) was evaluated using an atopic dermatitis mouse model to visually monitor disease course. Administration of iSG3caps improved skin lesions and decreased epidermal thickness. Underlying this effect is the ability of iSG3 to bind to and prevent phosphorylation of signal transducer and activator of transcription 6, thereby blocking the interleukin-4 signaling cascade mediated by binding of allergens to type 2 helper T cells. The results of our iSG3cap oral delivery experiments suggest that iSG3 may be useful for treating allergic diseases.

Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice.

BACKGROUND: Mucosal delivery of therapeutic proteins using genetically modified strains of lactic acid bacteria (gmLAB) is being investigated as a new therapeutic strategy. METHODS: We developed a strain of gmLAB, Lactococcus lactis NZ9000 (NZ-HO), which secretes the anti-inflammatory molecule recombinant mouse heme oxygenase-1 (rmHO-1). The effects of short-term continuous oral dosing with NZ-HO were evaluated in mice with dextran sulfate sodium (DSS)-induced acute colitis as a model of inflammatory bowel diseases (IBD). RESULTS: We identified the secretion of rmHO-1 by NZ-HO. rmHO-1 was biologically active as determined with spectroscopy. Viable NZ-HO was directly delivered to the colon via oral administration, and rmHO-1 was secreted onto the colonic mucosa in mice. Acute colitis in mice was induced by free drinking of 3 % DSS in water and was accompanied by an increase in the disease activity index score and histopathological changes. Daily oral administration of NZ-HO significantly improved these colitis-associated symptoms. In addition, NZ-HO significantly increased production of the anti-inflammatory cytokine interleukin (IL)-10 and decreased the expression of pro-inflammatory cytokines such as IL-1α and IL-6 in the colon compared to a vector control strain. CONCLUSIONS: Oral administration of NZ-HO alleviates DSS-induced acute colitis in mice. Our results suggest that NZ-HO may be a useful mucosal therapeutic agent for treating IBD.

Genetic mutation in Escherichia coli genome during adaptation to the murine intestine is optimized for the host diet.

Mammalian gut microbes colonize the intestinal tract of their host and adapt to establish a microbial ecosystem. The host diet changes the nutrient profile of the intestine and has a high impact on microbiota composition. Genetic mutations in Escherichia coli, a prevalent species in the human gut, allow for adaptation to the mammalian intestine, as reported in previous studies. However, the extent of colonization fitness in the intestine elevated by genetic mutation and the effects of diet change on these mutations in E. coli are still poorly known. Here, we show that notable mutations in sugar metabolism-related genes (gatC, araC, and malI) were detected in the E. coli K-12 genome just 2 weeks after colonization in the germ-free mouse intestine. In addition to elevated fitness by deletion of gatC, as previously reported, deletion of araC and malI also elevated E. coli fitness in the murine intestine in a host diet-dependent manner. In vitro cultures of medium containing nutrients abundant in the intestine (e.g., galactose, N-acetylglucosamine, and asparagine) also showed increased E. coli fitness after deletion of the genes-of-interest associated with their metabolism. Furthermore, the host diet was found to influence the developmental trajectory of gene mutations in E. coli. Taken together, we suggest that genetic mutations in E. coli are selected in response to the intestinal environment, which facilitates efficient utilization of nutrients abundant in the intestine under laboratory conditions. Our study offers some insight into the possible adaptation mechanisms of gut microbes.IMPORTANCEThe gut microbiota is closely associated with human health and is greatly impacted by the host diet. Bacteria such as Escherichia coli live in the gut all throughout the life of a human host and adapt to the intestinal environment. Adaptive mutations in E. coli are reported to enhance fitness in the mammalian intestine, but to what extent is still poorly known. It is also unknown whether the host diet affects what genes are mutated and to what extent fitness is affected. This study suggests that genetic mutations in the E. coli K-12 strain are selected in response to the intestinal environment and facilitate efficient utilization of abundant nutrients in the germ-free mouse intestine. Our study provides a better understanding of these intestinal adaptation mechanisms of gut microbes.

Expression of the immunoreactive buckwheat major allergenic storage protein in Lactococcus lactis.

Proteins from buckwheat (Fagopyrum esculentum) are strong allergens that can cause serious symptoms, including anaphylaxis, in patients with hypersensitivity. In this study, we successfully developed a modified lactic acid bacterial vector (pNSH) and a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced a major allergenic storage protein of buckwheat, Fagag1 (61.2 kDa, GenBank accession number AF152003), with or without a green fluorescent protein (GFP) tag. GFP fluorescence allows for rapid, simple, and accurate measurement of target protein expression by microscopy or fluorimetry. We describe a convenient method for production of rGFP-Fagag1 fusion and rFagag1 proteins with a good yield in an advantageous probiotic host. We found that in vitro treatment of splenocytes isolated from buckwheat crude protein-immunized mice with rFagag1 increased the expression of allergic inflammation cytokines such as IL-4, IL-13, and IL-17 F. Because it was less antigenic, rGFP-Fagag1 protein from NZ9000 might be of limited use; however, rFagag1 from NZ9000 evoked a robust response as measured by induction of IL-4 and IL-17 F expression levels. The observed allergic activity is indicative of a Th2 cell-mediated immune response and is similar to the effects induced by exposure to buckwheat crude protein. Our results suggest that expression of rFagag1 in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to buckwheat allergens.

Expression of a biologically active GFP-α(S1)-casein fusion protein in Lactococcus lactis.

In this study, we successfully developed a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced green fluorescent protein fused to α(S1)-casein (GFP-α(S1)Cas). A modified lactic acid bacterial vector (pNZ8148#2) was constructed by inserting genes for GFP and α(S1)-casein, a major cow's milk allergen, and the resulting vector, pNZ8148#2-GFP-α(S1)Cas, was applied to the expression of recombinant GFP-α(S1)Cas protein (rGFP-α(S1)Cas) in NZ9000. After inducing expression with nisin, the production of rGFP-α(S1)Cas was confirmed by confocal laser microscopic analysis, and the expression conditions were optimized based on fluorescent analysis and western blotting results. Moreover, the in vitro treatment of splenocytes isolated from α-casein (≥70 % α(S)-casein)-immunized mice with rGFP-α(S1)Cas resulted in increased IL-13 mRNA expression. The observed allergic activity is indicative of the Th2-cell mediated immune response and is similar to the effects induced by exposure to α-casein. Our results suggest that the expression of rGFP-α(S1)Cas in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to specific milk allergen.

Free Feeding of CpG-Oligodeoxynucleotide Particles Prophylactically Attenuates Allergic Airway Inflammation and Hyperresponsiveness in Mice.

CpG-oligodeoxynucleotides (CpG-ODNs) constitute an attractive alternative for asthma treatment. However, very little evidence is available from studies on the oral administration of CpG-ODNs in animals. Previously, we developed acid-resistant particles (named ODNcap) as an oral delivery device for ODNs. Here, we showed that free feeding of an ODNcap-containing feed prophylactically attenuates allergic airway inflammation, hyperresponsiveness, and goblet cell hyperplasia in an ovalbumin-induced asthma model. Using transcriptomics-driven approaches, we demonstrated that injury of pulmonary vein cardiomyocytes accompanies allergen inhalation challenge, but is inhibited by ODNcap feeding. We also showed the participation of an airway antimicrobial peptide (Reg3γ) and fecal microbiota in the ODNcap-mediated effects. Collectively, our findings suggest that daily oral ingestion of ODNcap may provide preventive effects on allergic bronchopulmonary insults via regulation of mechanisms involved in the gut-lung connection.

Oral priming with oligodeoxynucleotide particles from Lactobacillus rhamnosus GG attenuates symptoms of dextran sodium sulfate-induced acute colitis in mice.

Here, we investigated the effect of prophylactic oral treatment with carbonate apatite-based particles (ID35caps) containing Lactobacillus rhamnosus GG-derived immunostimulatory oligodeoxynucleotides (ID35) when used in mice with acute colitis. Mice were administered orally with control particles (carbonate apatite particles, Caps), ID35, or ID35caps for 2 days, and then were given free access to drinking water containing 3% (w/v) dextran sodium sulfate (DSS) for 5 days (Days 0-5) to induce acute colitis. Body weight change, fecal bleeding, and stool consistency were monitored and scored as a disease activity index (DAI) to assess symptoms of colitis. On Day 10, animals were euthanized and the colon length was measured to evaluate inflammatory tissue injury. Prophylactic oral treatment with ID35caps significantly suppressed DSS-induced elevation of the DAI score and shortening of the colon compared to the respective parameters in DSS-exposed mice treated with Cap or ID35. We conclude that oral priming with ID35caps attenuates symptoms and inflammatory colonic injury in a mouse model of DSS-induced acute colitis. This finding suggests that ID35caps may be a new oral agent for preventing intestinal inflammation.

Microbial therapeutics for acute colitis based on genetically modified Lactococcus lactis hypersecreting IL-1Ra in mice.

The increased incidence of inflammatory bowel disease (IBD) in Western and rapidly Westernizing developing countries poses a global pandemic threat. The development of affordable drugs for treating IBD worldwide is thus a priority. Genetically modified lactic acid bacteria (gmLAB) as microbial therapeutics are inexpensive protein producers suitable for use as carriers of protein to the intestinal mucosa. Here, we successfully constructed gmLAB hypersecreting interleukin 1 receptor antagonist (IL-1Ra). Oral administration of these gmLAB suppressed body weight reduction and exacerbation of the disease activity index score in mice with acute colitis and decreased the number of CD4+ IL-17A+ cells in the mesenteric lymph nodes. These data suggest that the gmLAB deliver IL-1Ra to the colon, where it inhibits IL-1 signaling. We thus developed a novel IBD therapeutic that blocks IL-1 signaling using a gmLAB protein delivery system. This system could be an inexpensive oral microbial therapeutic.

Nasally Administered Lactococcus lactis Secreting Heme Oxygenase-1 Attenuates Murine Emphysema.

Emphysema, a type of lung-destroying condition associated with chronic obstructive pulmonary disease (COPD), is an inflammatory lung disease mainly due to cigarette smoke exposure. As there is no curative therapy, prevention should be considered first by cessation of smoking to avoid exposure to oxidative stresses and inflammatory mediators. In addition, therapies involving antioxidative and/or anti-inflammatory agents such as heme oxygenase (HO)-1 are candidate treatments. We developed a new tool using genetically modified Lactococcus lactis to deliver recombinant HO-1 to the lungs. Using an elastase-induced emphysema model mimicking COPD, we evaluated the effect of nasally administered L. lactis secreting HO-1 (HO-1 lactis) on cellular and molecular responses in the lungs and further disease progression. Nasally administered HO-1 lactis resulted in (1) overexpression of HO-1 in the lungs and serum and (2) attenuation of emphysema progression evaluated both physiologically and morphologically. There was a transient 5-10% weight loss compared to baseline through trafficking to the lungs when administering 1.0 × 109 cells/mouse; however, this did not impact either survival or final body weight. These results suggest that delivering HO-1 using genetically modified L. lactis through the airways could be a safe and potentially effective therapeutic approach for COPD.

Oral Administration of Flavonifractor plautii, a Bacteria Increased With Green Tea Consumption, Promotes Recovery From Acute Colitis in Mice via Suppression of IL-17.

Flavonifractor plautii (FP) has been reported to participate in the metabolism of catechins in the human gut. However, there is limited information on the immune regulatory effects of this bacterium. We confirmed that the administration of green tea increases the abundance of FP in the gut microbiota and investigated the effect of FP in a mouse colitis model. Mice were orally administered FP for 10 consecutive days; colonic inflammation was evaluated daily on the basis of stool consistency, gross rectal bleeding, and body weight. In the dextran sodium sulfate model, FP-exposed animals exhibited lower levels of inflammation and strong inhibition of interleukin (IL)-17 signaling. Moreover, lipoteichoic acid from FP was identified as the active component mediating IL-17 suppression. Thus, oral administration of FP appears to modulate gut inflammation and represents a viable and inexpensive oral microbial therapeutic.

Oral Administration of Flavonifractor plautii Strongly Suppresses Th2 Immune Responses in Mice.

The bacterium Flavonifractor plautii (FP), which is found in human feces, has been reported to participate in catechin metabolism in the gut, but this bacterium's effects on immune function are unclear. We assessed the effect of oral administration of FP on the immune response in ovalbumin (OVA) -sensitized mice. We demonstrated that the FP treatment suppressed interleukin (IL)-4 in splenocytes and OVA-specific IgE production in serum from OVA-sensitized mice. Moreover, oral administration of FP augmented CD4+CD25+ T cells and CD103+CD11c+ DCs. In animals of the FP group, the proportion of FP was increased in the mesenteric lymph nodes (MLNs), as was the proportion of Deferribacteres in the cecum. Oral administration of FP may inhibit the Th2 immune response by incorporation into the MLNs and/or by inducing changes in the gut microbiota. Thus, FP may be useful in alleviating antigen-induced Th2 immune responses.

Construction of Genetically Modified Lactococcus lactis Producing Anti-human-CTLA-4 Single-Chain Fragment Variable.

Lactic acid bacteria are human commensal organisms that have immunomodulatory and metabolism-promoting effects. In addition, due to the increasing demand for biopharmaceuticals, genetically modified lactic acid bacteria (gmLAB) that produce recombinant proteins are expected to be used as microbial therapeutics and next-generation probiotics. In this study, we constructed a gmLAB strain that produces anti-human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) single-chain fragment variable (CTLA4scFv) for possible use in a cancer treatment strategy using gmLAB. CTLA-4, an immune checkpoint molecule, suppresses the anti-cancer immune response; thus, inhibition of CTLA-4 signaling is important in cancer therapy. In this study, we designed a CTLA4scFv composed of a heavy and light chain of the variable region from anti-human CTLA-4 antibody connected by a flexible peptide linker. CTLA4scFv was expressed using nisin controlled gene expression (NICE) system, a lactococcal inducible gene expression system, and the DNA sequence encoding CTLA4scFv was inserted downstream of the PnisA promoter of the gene expression vector pNZ8148#2. Furthermore, expression of recombinant CTLA4scFv was confirmed by Western blotting, and the immunoreactivity of recombinant CTLA4scFv against human CTLA-4 protein was examined using ELISA. We speculate that gmLAB producing bioactive CTLA4scFv will become an attractive approach for cancer treatment.

Lactobacillus ingluviei C37 from chicken inhibits inflammation in LPS-stimulated mouse macrophages.

Probiotics are growing alternatives to antibiotics, and can contribute to the prevention and treatment of diseases and enhance livestock production. Lactobacillus (L.) ingluviei is a novel probiotic species with growth-enhancement effects; however, this species remains poorly understood, and there have been (to our knowledge) no studies focusing on its immunological effects. Here, we isolated L. ingluviei C37 (LIC37) from chicken and evaluated the bacterium's immunomodulatory properties to explore its probiotic potential. Real-time quantitative PCR and ELISA showed that in vitro exposure of inflammation-stimulated mouse peritoneal macrophages to heat-killed LIC37 led to decreases in tumor necrosis factor-α and interleukin (IL)-6 levels and an increase in IL-10. These findings suggested that LIC37 exerts anti-inflammatory effects by modulating cytokine profiles. This species may be an attractive probiotic bacterial strain for use in animal production.