The motile marine bacterium, Vibrio vulnificus has a total of six flagellins. Flagellin is a structural component of flagellar filament in various locomotive bacteria and is the ligand of Toll-like receptor 5 (TLR5). TLRs, highly expressed on various types of cells including dendritic cells (DCs), recognize invading microorganisms and finally trigger host immune responses. In this study, we prepared all of six recombinant flagellin proteins and assessed the effect of six flagellins on IL-8 activation through TLR5 recognition. Although showed different activities, five out of the six flagellins stimulated significant IL-8 activation. We also investigated the immunomodulatory roles of Vv-FlaB, the crucial building block of V. vulnificus flagellar filament, on human dendritic cells. Treatment of immature DCs with Vv-FlaB resulted in an increased expression of co-stimulatory molecules and induced strong allo-T cell proliferative activities of the DCs. These results show that the Vv-FlaB may serve an epochal immune adjuvant possessing effective immunomodulatory activities.
Bacteria ; Dendritic Cells* ; Flagellin* ; Flow Cytometry ; Humans* ; Interleukin-8 ; Toll-Like Receptor 5 ; Vibrio vulnificus* ; Vibrio*

OBJECTIVETo study the association between single nucleotide polymorphisms(SNP) in toll-like receptors (TLR) 2 and 5 genes and the susceptibility to neonatal sepsis.

METHODSOne hundred and fourteen newborn infants who were diagnosed with clinical sepsis (case group) between May 2011 and January 2014 and 172 newborn infants without infection(control group) were enrolled in this study. The polymorphisms of TLR2 (rs5743708 and rs3804099) and TLR5 (rs5744105) were analyzed using a SNaPshot multiplex reaction to compare the genotypic and allelic frequencies between two groups. The relationship between TLR genotypes and susceptibility to sepsis was analyzed by logistic regression models.

RESULTSSignificant differences in genotypic frequencies of TLR2 rs3804099 (C/T) and TLR5 rs5744105 (C/G) were found between the two groups (P<0.05), but there was no significant difference in allelic frequencies of all the SNPs above between the two groups (P>0.05). The genotype on TLR2 rs5743708 was GG and no mutation was found in both groups. In regression models, birth weight (OR=3.065; P<0.05) and gestational age (OR=3.301; P<0.05) were closely associated with neonatal sepsis. Sex (OR=1.107, P>0.05), polymorphisms in rs3804099 (OR=0.876; P>0.05) and polymorphisms in rs5744105 (OR=0.820; P>0.05) genes were not risk factors for neonatal sepsis.

CONCLUSIONSTLR2 and 5 polymorphisms (rs5743708, rs3804099 and rs5744105) may not serve as the susceptible gene for sepsis in newborn infants.

Female ; Genetic Predisposition to Disease ; Humans ; Infant, Newborn ; Logistic Models ; Male ; Polymorphism, Single Nucleotide ; Sepsis ; genetics ; Toll-Like Receptor 2 ; genetics ; Toll-Like Receptor 5 ; genetics

PURPOSE: Recombinant subunit vaccines provide safe and targeted protection against microbial infections. However, the protective efficacy of recombinant subunit vaccines tends to be less potent than the whole cell vaccines, especially when they are administered through mucosal routes. We have reported that a bacterial flagellin has strong mucosal adjuvant activity to induce protective immune responses. In this study, we tested whether FlaB could be used as a fusion partner of subunit vaccine for tetanus. MATERIALS AND METHODS: We constructed fusion proteins consisted with tetanus toxin fragment C (TTFC), the nontoxic C-terminal portion of tetanus toxin, and a Toll-like receptor 5 agonist from Vibrio vulnificus (FlaB). Mice were intranasally administered with fusion protein and protective immune responses of the vaccinated mice were analyzed. RESULTS: FlaB-TTFC recombinant protein induced strong tetanus-specific antibody responses in both systemic and mucosal compartments and prolonged the survival of mice after challenge with a supra-lethal dose of tetanus toxin. CONCLUSION: This study establishes FlaB as a successful fusion partner for recombinant subunit tetanus vaccine applicable through mucosal route, and it further endorses our previous observations that FlaB could be a stable adjuvant partner for mucosal vaccines.
Animals ; Antibody Formation ; Flagellin* ; Mice ; Tetanus ; Tetanus Toxin* ; Tetanus Toxoid ; Toll-Like Receptor 5 ; Vaccines ; Vaccines, Subunit ; Vibrio vulnificus

Chronic inflammation has been considered an important risk factor for development of prostate cancer. Toll-like receptors (TLRs) recognize microbial moieties or endogenous molecules and play an important role in the triggering and promotion of inflammation. In this study, we examined whether expression of TLR4 and TLR5 was associated with progression of prostate transformation in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The expression of TLR4 and TLR5 was evaluated by immunohistochemisty in formalin-fixed paraffin-embedded prostate tissue from wild-type (WT) and TRAMP mice. Normal prostate tissue from WT mice showed strong expression of TLR4 and TLR5. However, TLR4 expression in the prostate tissue from TRAMP mice gradually decreased as pathologic grade became more aggressive. TLR5 expression in the prostate tissue from TRAMP mice also decreased in low-grade prostate intraepithelial neoplasia (PIN), high-grade PIN and poorly differentiated adenocarcinoma. Overall, our results suggest that decreased expression of TLR4 and TLR5 may contribute to prostate tumorigenesis.
Adenocarcinoma/etiology/*genetics ; Animals ; Cell Transformation, Neoplastic ; Disease Progression ; *Gene Expression Regulation, Neoplastic ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Prostatic Neoplasms/etiology/*genetics ; Toll-Like Receptor 4/*genetics/metabolism ; Toll-Like Receptor 5/*genetics/metabolism

INTRODUCTION: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atRA, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atRA regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atRA in immune cells. MATERIALS AND METHODS: This study examined whether atRA induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atRA-treated THP-1 cells. RESULTS: atRA at a concentration of 10 nM greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atRA stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. CONCLUSION: atRA induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atRA-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atRA and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers.
Apoptosis ; Binding Sites ; Blotting, Western ; Communicable Diseases ; Flagellin ; Humans ; Immunity, Cellular ; Interleukin-1 ; Leukemia ; NF-kappa B ; Pyrrolidines ; Retinoids ; Signal Transduction ; Thiocarbamates ; Toll-Like Receptor 5 ; Toll-Like Receptors ; Transcription Factors ; Tretinoin ; Vitamin A

BACKGROUND AND OBJECTIVES: Mammalian Toll-like receptors (TLR) participate in innate immune responses to microbial pathogens. Recent interest has been focused on the concept that TLR-induced innate responses can modulate subsequent adaptive immune responses. The objective of this study is to determine whether TLR 2 stimulation in vivo would modulate subsequent allergic responses in an Aspergillus fumigatus (Af) murine model of allergic rhinitis (AR). MATERIALS AND METHOD: Mice were sensitized via intraperitoneal injection with Af antigen and alum, and received a series of three daily intranasal Af antigen challenge. A TLR 2 agonist, PamCys was administrated intranasally one day before sensitization or one day before the first intranasal allergen challenge. Adaptive immune profiles and response to Af challenge were assessed. RESULTS: PamCys decreased the allergen induced nasal recruitment of eosinophils and interleukin (IL)-5 in nasal lavage fluids compared with mice treated with no PamCys. Serologic data revealed that PamCys downregulated Af-specific IgE in the sera of PamCys-treated mice. In addition, spleen cells from the PamCys-treated mice displayed attenuated Af-specific IL-4 and IL-5, but increased interferon (IFN)- and immunosuppressive IL-10. CONCLUSION: The present study demonstrate that TLR 2 agonist decreases allergic responses in this AR model by shifting T helper 2 (Th2) biased immune parameters towards Th1 dominance.
Animals ; Aspergillus fumigatus ; Bias (Epidemiology) ; Eosinophils ; Immunity, Innate ; Immunoglobulin E ; Injections, Intraperitoneal ; Interferons ; Interleukin-10 ; Interleukin-4 ; Interleukin-5 ; Interleukins ; Mice* ; Models, Animal ; Nasal Lavage Fluid ; Rhinitis* ; Spleen ; Toll-Like Receptor 2* ; Toll-Like Receptors*

PURPOSE: High-fat (HF) feeding induces hypothalamic leptin resistance via the activation of toll-like receptor 4 (TLR4). TLR4 deficiency confers resistance to diet-induced obesity. Udenafil, an anti-impotence drug, inhibits TLR4 in airway epithelial cells in vitro. In this study, we evaluated whether udenafil suppressed the hypothalamic expression of TLR4 and reduced body weight. MATERIALS AND METHODS: The hypothalamic expression of TLR4, phosphodiesterase 5 (PDE5), nuclear factor-κB (NF-κB), and myeloid differentiation primary response gene 88 (Myd88) was analyzed by real-time polymerase chain reaction after treating mice for 2 days with udenafil (0, 12, 120, or 600 µg/d). Furthermore, the hypothalamic expression of TLR4, pro-opiomelanocortin (POMC), and neuropeptide Y (NPY) was analyzed after 9 days' treatment with udenafil and/or leptin. We also measured body weight and food intake following 9 days of udenafil and/or leptin treatment in control- and HF-fed mice. RESULTS: Udenafil suppressed hypothalamic TLR4 mRNA expression dose-dependently. The changes were associated with decreased PDE5, NF-κB, and Myd88 expression. Udenafil treatment for 9 days reduced body weight and caloric intake in HF-fed mice. This may have been associated with the suppression of NPY expression that was elevated by HF feeding. POMC expression was not affected by udenafil. However, udenafil did not augment the effects of leptin on the reduction of body weight and caloric intake in HF-fed mice. CONCLUSIONS: These results suggested that udenafil reduced body weight by suppressing hypothalamic TLR4 mRNA expression in HF-fed mice and the combination effect of udenafil and leptin was additive rather than synergistic.
Animals ; Body Weight ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Eating ; Energy Intake ; Epithelial Cells ; Hypothalamus ; In Vitro Techniques ; Leptin ; Mice ; Neuropeptide Y ; Obesity ; Pro-Opiomelanocortin ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Toll-Like Receptor 4 ; Toll-Like Receptors

Postconditioning has been shown to protect the mouse brain from ischemic injury. However, the neuroprotective mechanisms of postconditioning remain elusive. We have found that toll-like receptor 5 (TLR5) plays an integral role in postconditioning-induced neuroprotection through Akt/nuclear factor kappa B (NF-κB) activation in cerebral ischemia. Compared to animals that received 30 min of transient middle cerebral artery occlusion (tMCAO) group, animals that also underwent postconditioning showed a significant reduction of up to 60.51% in infarct volume. Postconditioning increased phospho-Akt (p-Akt) levels and NF-κB translocation to the nucleus as early as 1 h after tMCAO and oxygen-glucose deprivation. Furthermore, inhibition of Akt by Akt inhibitor IV decreased NF-κB promoter activity after postconditioning. Immunoprecipitation showed that interactions between TLR5, MyD88, and p-Akt were increased from postconditioning both in vivo and in vitro. Similar to postconditioning, flagellin, an agonist of TLR5, increased NF-κB nuclear translocation and Akt phosphorylation. Our results suggest that postconditioning has neuroprotective effects by activating NF-κB and Akt survival pathways via TLR5 after cerebral ischemia. Additionally, the TLR5 agonist flagellin can simulate the neuroprotective mechanism of postconditioning in cerebral ischemia.
Animals ; Brain ; Brain Ischemia* ; Flagellin ; Immunoprecipitation ; In Vitro Techniques ; Infarction, Middle Cerebral Artery ; Mice* ; Neuroprotection ; Neuroprotective Agents ; NF-kappa B ; Phosphorylation ; Toll-Like Receptor 5

PURPOSE: Human papillomavirus (HPV) is a significant cause of cervical cancer-related deaths worldwide. Because HPV is a sexually transmitted mucosal pathogen, enhancement of antigen-specific mucosal immune response likely serves good strategy for vaccination. However, mucosal vaccines generally do not induce strong enough immune responses. Previously we proved that a bacterial flagellin, Vibrio vulnificus FlaB, induce strong antigen-specific immune responses by stimulating the Toll-like receptor 5. In this study, we tested whether FlaB could serve as an effective mucosal adjuvant for a peptide-based HPV preventive cancer vaccine. MATERIALS AND METHODS: Mice were intranasally administered with a mixture of FlaB and E6/E7 protective peptides in 5-day interval for a total of two times. Five-days after the last vaccination, cellular immune responses of the vaccinated mice were analyzed. Tumor growth was also observed after a subcutaneous implantation of TC-1 cells bearing E6/E7 antigens. RESULTS: Intranasal administration of the E6/E7 peptide mixture with FlaB elicited a strong antigen-specific cytotoxic T lymphocyte activity and antigen-specific interferon-gamma production from splenocytes and cervical lymph node cells. Furthermore, FlaB, as a mucosal adjuvant, conferred an excellent protection against TC-1 tumor challenge with high survival rates in E6/E7 immunized animals. CONCLUSION: These results indicate that FlaB can be a promising mucosal adjuvant for nasal HPV vaccine development.
Administration, Intranasal ; Animals ; Flagellin ; Humans ; Immunity, Cellular ; Immunity, Mucosal ; Immunization ; Interferon-gamma ; Lymph Nodes ; Lymphocytes ; Mice ; Peptides ; Survival Rate ; Toll-Like Receptor 5 ; Ursidae ; Vaccination ; Vaccines ; Vibrio vulnificus

PURPOSE: The house dust mite (HDM) is one of the most important sources of indoor allergens and a significant cause of allergic rhinitis and allergic asthma. Our previous studies demonstrated that Vibrio vulnificus flagellin B (FlaB) plus allergen as a co-treatment mixture improved lung function and inhibited eosinophilic airway inflammation through the Toll-like receptor 5 signaling pathway in an ovalbumin (OVA)- or HDM-induced mouse asthma model. In the present study, we fused the major mite allergen Derp2 to FlaB and compared the therapeutic effects of the Derp2-FlaB fusion protein with those of a mixture of Derp2 and FlaB in a Derp2-induced mouse asthma model. METHODS: BALB/c mice sensitized with Derp2 + HDM were treated with Derp2, a Derp2 plus FlaB (Derp2 + FlaB) mixture, or the Derp2-FlaB fusion protein 3 times at 1-week intervals. Seven days after the final treatment, the mice were challenged intranasally with Derp2, and airway responses and Derp2-specific immune responses were evaluated. RESULTS: The Derp2-FlaB fusion protein was significantly more efficacious in reducing airway hyperresponsiveness, lung eosinophil infiltration, and Derp2-specific IgE than the Derp2 + FlaB mixture. CONCLUSIONS: The Derp2-FlaB fusion protein showed a strong anti-asthma immunomodulatory capacity, leading to the prevention of airway inflammatory responses in a murine disease model through the inhibition of Th2 responses. These findings suggest that the Derp2-FlaB fusion protein would be a promising vaccine candidate for HDM-mediated allergic asthma therapy.
Allergens ; Animals ; Asthma ; Eosinophils ; Flagellin ; Immunoglobulin E ; Inflammation ; Lung ; Mice ; Mites ; Ovalbumin ; Pyroglyphidae ; Rhinitis, Allergic ; Therapeutic Uses ; Toll-Like Receptor 5 ; Vibrio vulnificus