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ABSTRACT: 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.
Applied Microbiology and Biotechnology 12/2012; · 3.42 Impact Factor
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ABSTRACT: 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.
Current Microbiology 03/2012; 64(6):569-75. · 1.82 Impact Factor
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ABSTRACT: We isolated cDNA encoding porcine nucleotide-binding domain-like receptor family, pryin domain containing 3 (NLRP3) from Peyer's patches. The complete nucleotide open reading frame of porcine NLRP3 contains 3108-bp encoding a deduced polypeptide of 1036-amino acid residues. The porcine NLRP3 amino acid sequence is more similar to the longest isoform of human than the mouse counterpart. The predicted amino acid sequence of porcine NLRP3 presented nine C-terminal leucine-rich repeat domains. In newborn swine, the expression of NLRP3 was detected at higher levels in spleen and mesenteric lymph nodes, while lower levels were observed in intestinal tissues. In adult swine, NLRP3 was strongly expressed in Peyer's patches and the mesenteric lymph nodes, and the expression level in the lower intestinal tissues was comparable to that in spleen. Toll-like receptor and nucleotide-binding domain ligands, as well as Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus gasseri, enhanced NLRP3 expression in gut-associated lymphoid tissues (GALT) of newborn and adult swine. Our results should aid in understanding the intestinal immunoregulatory mechanisms underlying NLRP3 activation and the priming ability of immunobiotic lactic acid bacteria in porcine GALT.
Veterinary Immunology and Immunopathology 12/2011; 144(3-4):410-6. · 2.08 Impact Factor
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Tomoyuki Shimazu,
Julio Villena,
Masanori Tohno,
Hitomi Fujie,
Shoichi Hosoya, Takeshi Shimosato,
Hisashi Aso,
Yoshihito Suda,
Yasushi Kawai,
Tadao Saito,
Seiya Makino,
Shuji Ikegami,
Hiroyuki Itoh,
Haruki Kitazawa
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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.
Infection and immunity 11/2011; 80(1):276-88. · 4.21 Impact Factor
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Shoichi Hosoya,
Julio Villena,
Tomoyuki Shimazu,
Masanori Tohno,
Hitomi Fujie,
Eriko Chiba, Takeshi Shimosato,
Hisashi Aso,
Yoshihito Suda,
Yasushi Kawai,
Tadao Saito,
Susana Alvarez,
Shuji Ikegami,
Hiroyuki Itoh,
Haruki Kitazawa
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ABSTRACT: ABSTRACT: 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 Peyer's 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.
Veterinary Research 11/2011; 42(1):111. · 4.06 Impact Factor
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Hitomi Fujie,
Julio Villena,
Masanori Tohno,
Kyoko Morie,
Tomoyuki Shimazu,
Hisashi Aso,
Yoshihito Suda, Takeshi Shimosato,
Noriyuki Iwabuchi,
Jin-Zhong Xiao,
Tomoko Yaeshima,
Keiji Iwatsuki,
Tadao Saito,
Muneo Numasaki,
Haruki Kitazawa
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ABSTRACT: A total of 23 strains of bifidobacteria taxonomically belonging to five species were tested for their potent immunomodulatory effect using a combination of two methods: the NF-κB-reporter assay using a toll-like receptor 2-expressing transfectant (HEK(pTLR2) system) and the mitogenic assay using porcine Peyer's patches immunocompetent cells. Among the four preselected strains from different immunomodulatory groups, Bifidobacterium breve MCC-117 was able to efficiently modulate the inflammatory response triggered by enterotoxigenic Escherichia coli (ETEC) in a porcine intestinal epithelial (PIE) cell line. Moreover, using PIE cells and swine Peyer's patches immunocompetent cell co-culture system, we demonstrated that the immunoregulatory effect of B. breve MCC-117 was related to the capacity of the strain to influence PIE and immune cell interactions, leading to the stimulation of regulatory T cells. The results suggested that bifidobacteria that express high activity in both the HEK(pTLR2) and the mitogenic assays may behave like potential anti-inflammatory strains. The combination of the HEK(pTLR2) system, the evaluation of mitogenic activity and PIE cells will be of value for the development of new immunologically functional foods and feeds that could prevent inflammatory intestinal disorders. Although our findings should be proven in appropriate experiments in vivo, the results of the present work provide a scientific rationale for the use of B. breve MCC-117 to prevent ETEC-induced intestinal inflammation.
FEMS Immunology & Medical Microbiology 06/2011; 63(1):129-39. · 2.44 Impact Factor
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ABSTRACT: Protein-energy malnutrition (PEM) is frequently associated with the occurrence of infection due to a decline in immune function. Here, an experiment was conducted with the objective of enhancing mucosal immunity by administration of Lactobacillus pentosus ONRIC b0240 (b240) in PEM model mice. Three groups of male C3H/HeN mice aged approximately 12 weeks were caged in groups of five or six and received various treatments. The mice were fed 4 (low-protein diet; PEM model), 20 (standard-protein diet), or 40% (high-protein diet) ovalbumin (OVA) with or without 0.05% b240. Five weeks later, all mice were sacrificed, and the organs were extracted for analysis of the immune response. Acute toxicity was not observed in this study. The addition of b240 showed no influence on body weight; however, body weight decreased with increasing protein level. Interestingly, intestinal total IgA was significantly increased (p<0.05) in all test diets with b240. The in vitro study showed that the number of B cells and type 2 helper T (Th2) cells were significantly increased in mouse spleen cells with b240 treatment, whereas no differences were found in the number of Th1 cells. b240 also has the ability to augment IgA and IgG production in mouse Peyer's patch cells. These results suggest that b240 enhances IgA production and helps recover the intestinal immune system in PEM model mice via augmentation of humoral immunity.
Journal of Agricultural and Food Chemistry 02/2011; 59(6):2646-51. · 2.82 Impact Factor
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ABSTRACT: We previously reported the strong immunostimulatory effects of a CpG oligodeoxynucleotide (ODN), designated MsST, from the lacZ gene of Streptococcus (S.) thermophilus ATCC19258. Here we show that 24h of stimulation with MsST in mouse splenocytes and peritoneal macrophages strongly induces expression of interleukin (IL)-33, a cytokine in the IL-1 superfamily. Other IL-1 superfamily members, including IL-1alpha, IL-1beta and IL-18, are down-regulated after 24h of stimulation of MsST. We also found that MsST-induced IL-33 mRNA expression is inhibited by the suppressive ODN A151, which can inhibit Toll-like receptor 9 (TLR9)-mediated responses. This is the first report to show that IL-33 can be induced by CpG ODNs. The strong induction of IL-33 by MsST suggests that it may be a potential therapeutic ODN for the treatment of inflammatory disease. The presence of a strong CpG ODN in S. thermophilus also suggests that the bacterium may be a good candidate as a starter culture for the development of new physiologically functional foods.
Biochemical and Biophysical Research Communications 02/2010; 394(1):81-6. · 2.48 Impact Factor
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ABSTRACT: Immunostimulatory sequences of oligodeoxynucleotides (ODNs), such as CpG ODNs, are potent stimulators of innate immunity. Here, we identified a strong immunostimulatory CpG ODN, which we named MsST, from the lac Z gene of Streptococcus (S.) thermophilus ATCC19258, and we evaluated its immune functions. In in vitro studies, MsST had a similar ability as the murine prototype CpG ODN 1555 to induce inflammatory cytokine production and cell proliferation. In mouse splenocytes, MsST increased the number of CD80+CD11c+and CD86+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells. We also analyzed the effects of MsST on the expression of regulatory cytokines by real-time quantitative PCR. MsST was more potent at inducing interleukin-10 expression than the ODN control 1612, indicating that MsST can augment the regulatory T cell response via Toll-like receptor 9, which plays an important role in suppressing T helper type 2 responses. These results suggest that S. thermophilus, whose genes include a strong Immunostimulatory sequence-ODN, is a good candidate for a starter culture to develop new physiologically functional foods and feeds.
Animal Science Journal 10/2009; 80(5):597-604. · 0.86 Impact Factor
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ABSTRACT: Lymphotoxin-beta (LT-beta) has been suggested to be a regulator of secondary lymphoid structure development. In the present study, we isolated porcine LT-beta (poLT-beta) 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-beta protein. Expression of LT-beta mRNA in various swine tissues was analyzed by real-time PCR, and it was found to be higher in the ileal Peyer's 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-beta 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-beta via toll-like receptors. LT-beta may therefore be useful for studying the development of the intestinal immune system at parturition in both swine and humans.
Biochemical Genetics 02/2009; 47(1-2):126-36. · 0.86 Impact Factor
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ABSTRACT: Recent interest has focused on the importance of intestinal immunity for the host defense, but to date, not much is known about the underlying mechanisms. The toll-like receptor (TLR) family plays an important role in host defense through recognizing bacterial pathogen-associated molecular patterns. Our recent research on the physiological function of food products has investigated the immunoregulatory effects of probiotic lactic acid bacteria (LAB) via TLR. Studies of swine, which often substitute for a human model, have demonstrated intestinal immunoregulation by the probiotic LAB mediated by TLR in the gut. On the basis of our study, efforts have also been made to develop a molecular immunoassay system for probiotic LAB and find novel immunostimulatory DNA sequences from probiotics and high potential immunobiotic LAB strains via TLR signaling. These findings may provide important clues at the molecular level on TLR signal transduction pathways and recognition mechanisms for the ligands. They also provide impetus to further delineate the activation mechanism of the innate immune response. In addition to identifying immunoregulatory factor immunogenics from LAB, a better understanding of intestinal immune regulation through cytokine networks holds out promise for basic food immunology research and the development of immunobiotic foods to prevent specific diseases.
Animal Science Journal 10/2007; 79(1):11 - 21. · 0.86 Impact Factor
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ABSTRACT: Proteins in the milk release biologically active peptides upon enzymatic digestion. In the present study, we report the identification of novel monocyte/macrophage chemotactic peptides derived from enzymatically digested bovine beta-casein, a casein family member that is a major constituent of milk. Beta-casein fragments generated by actinase E showed potent chemotactic activity for human and mouse monocytes/macrophages, but not neutrophils, T lymphocytes or dendritic cells. The fragment-induced migration of human monocytes was inhibited by pertussis toxin and was not desensitized by a variety of known chemoattractants, suggesting that the digests activate a unique G protein-coupled receptor(s). The digests were further fractionated and purified to yield 3 small peptides. One peptide Q1 designated as "beta-casochemotide-1" with the amino acid sequence of YPVEP (f114-118 of beta-casein) induced high levels of macrophage chemotaxis. It also promoted calcium mobilization in macrophages, another indication of cell activation. Our study suggests that biologically active peptides released by actinase-digested milk beta-casein may promote innate host immune responses by inducing macrophage migration and activation.
International Immunopharmacology 10/2007; 7(9):1150-9. · 2.38 Impact Factor
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ABSTRACT: Toll-like receptor 2 (TLR2) is a receptor for a variety of microbial components, and it also mediates activation signals in the cell relating to the innate immune system. In order to evaluate the precise molecular immunoregulation by various strains of lactic acid bacteria (LAB) via TLR2, the swine TLR2 (sTLR2)-expressing transfectant was constructed using human embryonic kidney (HEK) 293 cells. It is demonstrated that intact immunobiotic LAB can induce immune responses through TLR2, and that different nuclear factor-κB (NF-κB) activities of various strains can be accurately detected by sTLR2-expressing HEK293 cells. Furthermore, cellular activation of NF-κB via TLR2 is reflected in enhanced binding and uptake of LAB. The sTLR2-expressing HEK293 cells were also useful for characterizing the expression pattern of type I helper T (Th1) and type II helper T (Th2) cytokines by the stimulation of immunobiotic LAB. These results suggest that sTLR2-expressing HEK293 cells may be useful in certain molecular immunoassay systems for producing new physiologically functional foods with intestinal immunomodulatory abilities, such as the maintenance of Th1/Th2 polarization.
Animal Science Journal 03/2007; 78(2):195 - 205. · 0.86 Impact Factor
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ABSTRACT: The immunostimulatory oligodeoxynucleotide (ODN) BL07 (5'-GCGTCGGTTTCGGTGCTCAC-3') was identified from the genomic DNA of the probiotic strain Bifidobacterium longum BB536. ODN BL07 stimulated B-lymphocyte proliferation and induced interleukin-12 (IL-12) production in macrophage-like J774.1 cells. ODNs BL07 and BL07S (modified with phosphorothioate backbone) significantly inhibited immunoglobulin E (IgE) production and stimulated interferon-gamma (IFN-gamma) and IL-12 production, but did not affect IL-4 secretion in murine splenic cells of ovalbumin-primed BALB/c mice. These ODNs also significantly inhibited production of IgE in purified murine B cells in the presence of IL-4 and anti-CD40. The results suggest the potential of ODNs BL07 and BL07S in preventing IgE-related immune responses and the possible involvement of ODN BL07 in the antiallergic efficacy of B. longum BB536.
FEMS Immunology & Medical Microbiology 05/2006; 46(3):461-9. · 2.44 Impact Factor
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ABSTRACT: 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 JCM1131(T). 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-kappaB 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.
Cellular Microbiology 04/2006; 8(3):485-95. · 5.46 Impact Factor
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ABSTRACT: Toll-like receptor 2 (TLR2) has been shown to mediate cell signaling in response to microbial cell wall components, such as peptidoglycan, lipoteichoic acid, microbial lipoprotein, and zymosan. In this study, we cloned the swine TLR2 and used it to transfect Chinese hamster ovary K-1 cells. We demonstrated that the swine TLR2-expressing transfectant can bind not only zymosan from yeast cell wall components but also intact lactic acid bacteria, resulting in the activation of nuclear factor-kappaB. These findings suggest that the swine TLR2-expressing transfectant can be very useful for the primary screening of immunobiotic microorganisms.
FEMS Immunology & Medical Microbiology 07/2005; 44(3):283-8. · 2.44 Impact Factor
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Masanori Tohno, Takeshi Shimosato,
Haruki Kitazawa,
Shinichiro Katoh,
Iliyan Dimitrov Iliev,
Toshiro Kimura,
Yasushi Kawai,
Kouichi Watanabe,
Hisashi Aso,
Takahiro Yamaguchi,
Tadao Saito
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ABSTRACT: The Toll-like receptor (TLR) 2 binds a wide variety of microbial cell wall components. In this study, we investigated the expression pattern of TLR2 in adult swine gut-associated lymphoid tissues using real-time quantitative PCR, Western blotting, immunohistochemistry, and flow cytometric analysis. The mRNA for TLR2 was preferentially expressed in the mesenteric lymph nodes (MLNs) and Peyer's patches (Pps) of adult swine. Expression in these two tissues was approximately 15- and 9-fold higher than that of spleen, respectively. Western blotting further confirmed that the TLR2 protein was highly expressed in the MLNs and Pps. Interestingly, TLR2-expressing cells were found not only in immune cells, such as T cells and B cells, but also in membranous (M) cells. In addition, double immunostaining for TLR2 and cytokeratin 18 revealed that TLR2 was strongly expressed not only in the cytoplasm but also in the apical membrane of the pocket-like M cells. These results indicate that TLR2 on the MLNs and Pps enable the host defense to respond to a variety of cell wall components. Furthermore, the potential function of TLR2 as a pattern recognition receptor and its cellular distribution suggest that TLR2 plays an important role in ligand-specific transcytosis and transport in M cells.
Biochemical and Biophysical Research Communications 06/2005; 330(2):547-54. · 2.48 Impact Factor
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ABSTRACT: The precise distribution and expression of Toll-like receptor (TLR) 9 in gut-associated lymphoid tissues (GALTs) has not been elucidated. In this study, we investigated the expression pattern of TLR9 in adult and neonatal swine GALTs by real-time quantitative PCR, western blot, confocal laser microscopy and flow cytometric analysis. The swine TLR9 gene was preferentially expressed in adult Peyer's patches (Pps) and mesenteric lymph nodes (MLNs), which contained approximately three times higher TLR9 than the spleen. Other tissues exhibited only weak expression of TLR9. In neonatal swine, elevated expression of TLR9 was detected only in MLNs. We firstly showed that highly expressive (TLR9(+)) cells were formed in Pps and MLNs. In addition, TLR9(+) cells were present not only in immune cells such as dendritic cells and B cells but also in follicle-associated epithelia (FAE) including membranous cells (M cells) in Pps. These results suggest that Pps and MLNs provide the host defense with the ability to respond to a variety of bioactive oligonucleotides (ODNs) from bacteria at a conductive site of initial immune responses.
Immunology Letters 05/2005; 98(1):83-9. · 2.53 Impact Factor
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ABSTRACT: 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-gamma) and tumour necrosis factor alpha (TNF-alpha) mRNA expression and IFN-gamma/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.
Cellular Microbiology 04/2005; 7(3):403-14. · 5.46 Impact Factor
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ABSTRACT: Toll-like receptor 9, which is expressed on the surface of antigen presenting cells and which was recently identified in the cytoplasmic follicle, recognizes bacterial CpG oligodeoxynucleotides (ODNs), resulting in the induction of a potent immune response. However, in our previous study, we found that TLR9 potentially recognizes not only CpG ODN but also non-CpG ODN such as AT ODN. Therefore, in the present study, to investigate this possibility, we elucidated the effects of AT ODN on T(H)-1, T(H)-2 type cytokine induction via TLR9 by real-time quantitative PCR analysis and ELISA of the swine TLR9 transfectant. The results demonstrated that the T(H)-1 type cytokines such as interleukin (IL)-12p70 and interferon (IFN)-gamma were strongly induced by AT ODN compared to the unexposed controls, while T(H)-2 type cytokines were not induced. These results indicate that the AT ODN can augment the T(H)-1 immune response, which plays an important role in prevention of allergic responses. Moreover, the swine TLR9 transfectant demonstrated its usefulness for evaluation of immunostimulation by bacterial DNA through the detection of T(H)-1, T(H)-2 type cytokine induction via TLR9 signaling.
Biochemical and Biophysical Research Communications 02/2005; 326(4):782-7. · 2.48 Impact Factor
