OBJECTIVE: To investigate the effect of aquaporin 5 (AQP5) on Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor κB (NF-κB) signaling pathway in Sjögren syndrome (SS) rats. METHODS: The SS gene expression data sets GSE406611 and GSE84844 were extracted from the Gene Expression Omnibus (GEO), and the AQP5 mRNA expression was analyzed by R software. The rat SS model was constructed. The successfully modeled rats were divided into SS group, SS+NC group, and SS+pc group, 10 rats in each group; and 10 rats were set as Normal group. The rats in the SS+NC group were injected with 10 μg of rno-pcDNA3.1-AQP5-NC at the submandibular gland, subcutaneously every day for 28 days. The rats in the SS+pc group were injected with 10 μg of rno-pcDNA3.1-AQP5 at the submandibular gland, subcutaneously every day for 28 days. The enzyme-linked immunosorbent assay (ELISA) kit was used to detect the content of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the serum. High-throughput sequencing was used to identify the target genes. Quantitative real-time PCR (qPCR) and Western blot were used to detect the mRNA and protein expressions of AQP5, TLR4, MyD88, and NF-κB in the rat submandibular gland tissue. RESULTS: In the SS dataset GSE406611 and GSE84844, the mRNA expression of AQP5 in SS was significantly reduced. Compared with the Normal group, the content of TNF-α and IL-1β in the serum, the mRNA and protein expressions of TLR4, MyD88, and NF-κB in the SS group were significantly increased, the mRNA and protein expressions of AQP5 were significantly decreased. After overexpression of AQP5, the content of TNF-α and IL-1β in the serum, the mRNA and protein expressions of TLR4, MyD88, and NF-κB in the SS+pc group were significantly decreased, the mRNA and protein expressions of AQP5 were significantly increased. The differences were statistically significant (all P < 0.05). CONCLUSION The expression of AQP5 is involved in the progression of SS. Increasing the expression of AQP5 can significantly inhibit inflammatory stress and reduce the pathological damage of submandibular gland tissue. This may be related to the inhibition of TLR4/MyD88/NF-κB conduction.
Animals ; Toll-Like Receptor 4/genetics* ; Myeloid Differentiation Factor 88/genetics* ; Aquaporin 5/metabolism* ; Sjogren's Syndrome/genetics* ; Signal Transduction ; NF-kappa B/metabolism* ; Rats ; Rats, Sprague-Dawley ; Interleukin-1beta/metabolism* ; Female

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

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: Invariant natural killer T (iNKT) cells play a critical role in the pathogenesis of asthma. We previously reported the association between circulating Th2-like iNKT cells and lung function in asthma patients and the suppressive effect of Toll-like receptor 5 ligand flagellin B (FlaB) on asthmatic in a mouse model. Thus, we investigated whether FlaB modulates the function of circulating iNKT cells in asthmatic patients. METHODS: Peripheral blood mononuclear cells (PBMCs) were treated with FlaB, and the secreted and intracellular cytokines of iNKT cells were evaluated by using ELISA and flow cytometry, respectively, following stimulation with alpha-galactosylceramide. Foxp3+ iNKT cells were also measured. To determine the effect of FlaB-treated dendritic cells (DCs) on iNKT cells, we co-cultured CD14+ monocyte-derived DCs and T cells from patients with house dust mite-sensitive asthma and analyzed intracellular cytokines in iNKT cells. RESULTS: A reduction of IL-4 and IL-17 production by iNKT cells in PBMCs after FlaB treatment was alleviated following blocking of IL-10 signaling. A decrease in the frequencies of IL-4+ and IL-17+ iNKT cells by FlaB-treated DCs was reversed after blocking of IL-10 signaling. Simultaneously, an increase in Foxp3+ iNKT cells induced by FlaB treatment disappeared after blocking of IL-10. CONCLUSIONS: FlaB may inhibit Th2- and Th17-like iNKT cells and induce Foxp3+ iNKT cells by DCs via an IL-10-dependent mechanism in asthmatic patients. In patients with a specific asthma phenotype associated with iNKT cells, FlaB may be an effective immunomodulator for iNKT cell-targeted immunotherapy.
Animals ; Asthma* ; Cytokines ; Dendritic Cells* ; Dust ; Enzyme-Linked Immunosorbent Assay ; Flagellin* ; Flow Cytometry ; Humans ; Immunotherapy ; Interleukin-10 ; Interleukin-17 ; Interleukin-4 ; Lung ; Mice ; Natural Killer T-Cells* ; Phenotype ; T-Lymphocytes ; Toll-Like Receptor 5

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

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

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

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-kappaB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation.
2',5'-Oligoadenylate Synthetase/genetics/metabolism ; Animals ; Cell Line ; *Cloning, Molecular ; Ducks ; Gene Expression Regulation/*physiology ; Humans ; Immunity, Innate ; Myeloid Differentiation Factor 88/genetics/metabolism ; Myxovirus Resistance Proteins/genetics/metabolism ; Newcastle Disease/metabolism ; Newcastle disease virus/classification ; RNA, Messenger/genetics/metabolism ; Species Specificity ; Toll-Like Receptor 5/genetics/*metabolism

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