Toll-Like Receptor 3 Agonist Protection against Experimental Francisella tularensis Respiratory Tract Infection

We investigated whether Toll-like receptor 3 (TLR3) stimulation would protect the host from inhaled Francisella tularensis. TLR3 is expressed by respiratory epithelial cells and macrophages and can be activated by a synthetic double-stranded RNA ligand called polyinosine-polycytosine [poly(I:C)]. Thus, we evaluated poly(I:C) as a novel treatment against inhaled F. tularensis. In vivo, BALB/c mice intranasally (i.n.) treated with poly(I:C) (100 μg/mouse) 1 h before or after Schu 4 or LVS (100 CFU) i.n. challenge showed that poly(I:C) treatment significantly reduced bacterial load in the lungs (P < 0.05). Bronchoalveolar lavage from poly(I:C)-treated mice alone or combined with F. tularensis infection significantly increased cytokine secretion and enhanced neutrophil influx to lung tissues. Poly(I:C) responses were transient but significantly prolonged the survival of treated mice after i.n. F. tularensis challenge relative to mock treated animals. This prolonged survival providing a longer window for initiation of levofloxacin (LEVO) treatment (40 mg/kg). Animals treated with poly(I:C), challenged with F. tularensis, and then treated with LEVO 5 days later had 100% survival relative to 0% survival in animals receiving LEVO alone. Mechanistically, poly(I:C) given to human monocyte-derived macrophages before or after Schu 4 or LVS challenge (multiplicity of infection, 20:1) had significantly reduced intracellular bacterial replication (P < 0.05). These data suggest that poly(I:C) may represent a potential therapeutic agent against inhaled F. tularensis that prolongs survival and the opportunity to initiate standard antibiotic therapy (i.e., LEVO).Inhalation is likely to be one of the primary routes by which a bioweapon will be delivered to a target population. Francisella tularensis is a potential bioweapon because it can be aerosolized due to its inherently hardy nature, and less than 20 inhaled organisms can be detrimental to the host (8, 10, 28). F. tularensis, the etiologic agent of tularemia, is a small, Gram-negative nonmotile coccus and a facultative intracellular bacterium (16, 36). There are two major subspecies of F. tularensis; one is designated type A and includes strains that induce aggressive pathologies in the host and can result in pulmonary tularemia causing death if not treated (18, 37). The commonly studied virulent type A strain, Schu 4, was isolated originally from a human case of tularemia (22, 31, 38). The type B subspecies of F. tularensis causes a milder disease in humans than do type A strains. The only vaccine against tularemia known at this time was derived from subspecies type B and is called the live vaccine strain (LVS) (3, 8, 19, 31). Interestingly, most animal modeling of F. tularensis infection has used LVS-infected mice because it mimics the human disease caused by type A strains (6, 7, 9, 12). The immunological efficacy of LVS in humans is not known; vaccination with LVS does not provide complete protection against the virulent type A strains of F. tularensis (7, 19). As a result, alternative intervention strategies and vaccines need to be developed.Ideally, vaccines against bioweapons will be established to protect the general population limiting the impact of such terroristic acts. Until such vaccines are available and widely distributed, alternate methods of broad range protection must be investigated. One interesting strategy is to engender innate immune resistance against mucosal pathogens (13, 17, 24). We have investigated the potential of Toll-like receptor (TLR) agonists recognized by TLRs highly expressed by respiratory epithelial cells. Specifically, polyinosine-polycytosine [poly(I:C)] is a synthetic double-stranded RNA analog that stimulates TLR3 triggering the induction of the host innate immune response including as RANTES, gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-6 (11, 17, 23, 26).Poly(I:C) can be delivered easily by a nose spray, is cheap to manufacture, and could be offered as an over-the-counter product unlike antibiotics. Importantly, the kinetics of cytokine secretion after poly(I:C) administration showed a transient response and offered no indication of toxicity, even with repeated use in our previous study (17). We therefore examined poly(I:C) as a topical treatment for a potential F. tularensis aerosol release. Theoretically, intranasal (i.n.) poly(I:C) could engender an innate immune response against F. tularensis, providing an extended period of resistance before an antibiotic, such as levofloxacin (LEVO), can be administered. LEVO belongs to the group of antibiotics known as fluoroquinolones and has been used to treat respiratory infections such as tularemia (1, 21, 25).Recently, we established that genital application of poly(I:C) protected against lethal HSV-2 challenge in mice (17). We have extended these findings by applying poly(I:C) to the respiratory mucosa testing the hypothesis that nucleic acid-based TLR agonists may prove to be useful prophylactic and possibly therapeutic measures against select agent respiratory infections including F. tularensis. Because F. tularensis suppresses the innate immune response (2, 4, 29, 39), the host does not detect and/or respond to the organism for approximately 48 to 72 h after F. tularensis infection (2; T. D. Eaves-Pyles, unpublished data). This large gap between the time of infection and host detection of the organism limits the development of an adequate immune response against F. tularensis. As such, we hypothesized that poly(I:C) would enhance the host''s response prior to or soon after F. tularensis exposure. Our in vivo and in vitro studies show that mice treated 1 h before or 1 h after the administration of poly(I:C) had significantly less bacteria in their lungs, increased neutrophil infiltration to the lung, and extended survival after LVS or Schu 4 infection. Moreover, mice treated with poly(I:C) (1 h after Schu 4 infection), followed by LEVO administration 5 days later, were fully protected from lethal outcomes. Corresponding to these in vivo studies, we show that poly(I:C)-treated human monocyte-derived macrophages (MDM) secreted high levels of specific cytokines and engendered enhanced intracellular bacterial killing after LVS and Schu 4 exposure compared to untreated animals.     

Mutations in Toll-Like Receptor 3 Are Associated with Elevated Levels of Rotavirus-Specific IgG Antibodies in IgA-Deficient but Not IgA-Sufficient Individuals

Double-stranded RNA (dsRNA) triggers immune-mediated responses through toll-like receptor 3 (TLR3), which is involved in innate antiviral defense. Low expression of TLR3 was recently suggested to contribute to susceptibility to rotavirus infection. Thus, we investigated the role of two TLR3 polymorphisms (rs3775291 and rs5743305), both of which resulted in reduced protein function or expression, in healthy blood donors and IgA-deficient (IgAD) individuals. These polymorphisms were associated with elevated rotavirus-specific IgG titers in IgAD individuals but not in healthy individuals. Thus, we propose that TLR3 signaling does not contribute to the rotavirus-specific antibody response in IgA-sufficient individuals, whereas it is associated with elevated antibody titers in IgAD individuals.     

Differential Expression of Toll-Like Receptor Signaling Pathway Is Associated with Microscopic Polyangiitis in Peripheral Blood Neutrophils

Constitutive or excessive activation of Toll-like receptor (TLR) signaling pathway can disrupt the body’s immune tolerance to autoantigen, thus promoting the development of autoimmune disease. However, the expression profile of TLR signaling pathway in peripheral blood neutrophils in the pathogenesis of microscopic polyangiitis (MPA) remains unclear. Thus, improved understanding of the pathobiology of this disease may aid in the development of therapeutic targets for patients with MPA. In the present study, we assessed the expression of TLR signaling pathway-related genes in peripheral blood neutrophils in patients with MPA. PCR array analysis was performed on 20 patients with MPA and 12 healthy controls. Gene expression pro?le was performed using the human TLR for autoimmunity and inflammation PCR array of Genecopoeia, containing 84 genes related to TLR signaling pathway and six house-keeping genes. We then used quantitative real-time PCR to validate the array test. The array results identified 13 upregulated genes and 5 genes which were downregulated. The resulting qRT-PCR was consistent with the findings by PCR array. Our results suggest that peripheral blood neutrophils display changes in the expression of TLR signaling pathway-related genes associated with the pathogenesis of microscopic polyangiitis.     

Toll-Like Receptor 2 Arg677Trp Polymorphism Is Associated with Susceptibility to Tuberculosis in Tunisian Patients

Toll-like receptor 2 (TLR2) is critical in the immune response to mycobacteria. Herein, we report that the frequency of a human TLR2 Arg677Trp polymorphism (C2029T nucleotide substitution) in tuberculosis patients in Tunisia is significantly higher than in healthy controls (P < 0.0001). This finding suggests that this polymorphism could be a risk factor for tuberculosis.     

Berberine Ameliorates Collagen-Induced Arthritis in Rats Associated with Anti-inflammatory and Anti-angiogenic Effects

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammation and joint destruction. In this study, we explored the effect of berberine on rats with bovine type II collagen-induced arthritis (CIA), an animal model for RA. Following treatment, berberine attenuates arthritic scores and suppresses collagen–specific immune responses in CIA rats. Compared with the un-treated CIA group, berberine reversed pathological changes, which showed a significant improvement in synovial hyperplasia and inflammatory infiltration. The expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-17 and vascular endothelial growth factor (VEGF) were obviously reduced in the sera of berberine-treated rats (all P?VEGF and CD34 (all P?P?相似文献   

Toll-Like Receptor 9-Mediated Inflammation Triggers Alveolar Bone Loss in Experimental Murine Periodontitis

Chronic periodontitis is a local inflammatory disease induced by a dysbiotic microbiota and leading to destruction of the tooth-supporting structures. Microbial nucleic acids are abundantly present in the periodontium, derived through release after phagocytic uptake of microbes and/or from biofilm-associated extracellular DNA. Binding of microbial DNA to its cognate receptors, such as Toll-like receptor 9 (TLR9), can trigger inflammation. In this study, we utilized TLR9 knockout (TLR9^−/−) mice and wild-type (WT) controls in a murine model of Porphyromonas gingivalis-induced periodontitis and report the first in vivo evidence that TLR9 signaling mediates the induction of periodontal bone loss. P. gingivalis-infected WT mice exhibited significantly increased bone loss compared to that in sham-infected WT mice or P. gingivalis-infected TLR9^−/− mice, which were resistant to bone loss. Consistent with this, the expression levels of interleukin 6 (IL-6), tumor necrosis factor (TNF), and receptor-activator of nuclear factor kappa B ligand (RANKL) were significantly elevated in the gingival tissues of the infected WT mice but not in infected TLR9^−/− mice compared to their levels in controls. Ex vivo studies using splenocytes and bone marrow-derived macrophages revealed significantly diminished cytokine production in TLR9^−/− cells relative to the cytokine production in WT cells in response to P. gingivalis, thereby implicating TLR9 in inflammatory responses to this organism. Intriguingly, compared to the cytokine production in WT cells, TLR9^−/− cells exhibited significantly decreased proinflammatory cytokine production upon challenge with lipopolysaccharide (LPS) (TLR4 agonist) or Pam3Cys (TLR2 agonist), suggesting possible cross talk between TLR9, TLR4, and TLR2. Collectively, our results provide the first proof-of-concept evidence implicating TLR9-triggered inflammation in periodontal disease pathogenesis, thereby identifying a new potential therapeutic target to control periodontal inflammation.     

TLR4信号转导通路在肿瘤中的作用

慢性炎症与恶性肿瘤密切相关,Toll样受体4(TLR4)在肿瘤中的广泛表达提示其在慢性炎症致瘤机制中发挥重要作用.活化肿瘤细胞TLR4不仅促进肿瘤的生成和转移,而且参与肿瘤的免疫逃逸.另一方面,免疫佐剂又通过激活免疫细胞的TLR4信号产生抗肿瘤免疫.因此,TLR4在肿瘤中起着双刃剑的作用.     

Single Nucleotide Polymorphisms in the Toll-Like Receptor 3 and CD44 Genes Are Associated with Persistence of Vaccine-Induced Immunity to the Serogroup C Meningococcal Conjugate Vaccine

The rate of decay of antibody concentration following serogroup C meningococcal (MenC) polysaccharide-protein conjugate vaccination varies between individuals. This depends partly on vaccination age but may be influenced by human genetics. We studied 721 single nucleotide polymorphisms (SNPs) across 131 candidate genes in a first cohort of 905 Caucasians (11 to 21 years old; mean time after vaccination, 4.9 years) and 30 SNPs across 17 genes in a replication study using 155 children, aged 6 to 12 years (mean time after vaccination, 6.7 years), and 196 infants (1 year old; mean time after vaccination, 8 months). Individuals were classified as responders or nonresponders for total MenC IgG concentration and MenC serum bactericidal antibody (SBA) measurements. Associated genes were examined further for quantitative outcome measures. Fifty-nine SNPs in 37 genes were associated with IgG persistence (adjusted for age at measurement), and 56 SNPs in 36 genes were associated with SBA persistence (adjusted for age at measurement and vaccine used). Three SNPs each within the Toll-like receptor 3 (TLR3) (rs3775291, rs3775292, and rs5743312) and CD44 (rs11033013, rs353644, and rs996076) genes were associated with IgG (adjusted for age at measurement) or SBA (adjusted for age at measurement and vaccine used) persistence in the initial genetic study (P, 0.02 to 0.04). Single SNPs within the TLR3 (rs7657186) (P = 0.004 [unadjusted]) and CD44 (rs12419062) (P = 0.01 [unadjusted]) genes were associated with IgG persistence in the replication study. These results suggest that genetic polymorphisms in the TLR3 and CD44 genes are associated with the persistence of the immune response to MenC vaccines 1 to 6 years after vaccination.     

Single Nucleotide Polymorphisms of Toll-Like Receptor 7 and Toll-Like Receptor 9 in Hepatitis C Virus Infection Patients from Central China

Purpose

To analyze the correlation of polymorphisms of toll-like receptor 7 (TLR7) (rs179009) and toll-like receptor 9 (TLR9) (rs187084) in hepatitis C virus (HCV) infections in the Han population.

Materials and Methods

The genotypes of TLR7IVS2-151 in HCV infection were detected by Sanger sequencing using polymerase chain reaction-restriction fragment length polymorphism to determine the TLR9 T-1486C single nucleotide polymorphisms (SNP) for all enrolled patients.

Results

We found no significant difference between males with spontaneous clearance of HCV versus those chronically infected [χ²=2.71, p=0.10, odd ratios (OR)=0.58, 95% confidence interval (CI) 0.31-1.11]. However, significant differences were found for the distribution of TLR7 (rs179009) in females (χ²=9.46, p=0.01). In females, a significant difference was also found between chronic hepatitis C and those with spontaneous clearance of HCV in terms of TLR7 IVS2-151G/A allele frequencies (χ²=9.50, p=0.00, OR=0.46, 95% CI 0.28-0.75). In HCV-infected patients, no significant association was found between the frequency of TLR9 genotypes and alleles.

Conclusion

The site of TLR7 IVS2-151 (rs179009) G/A may be a factor for susceptibility of chronic HCV in the female Han population. TLR9T-1486C (rs18084) SNP may not play a major role in HCV infection. However, individual risk profiles for HCV infection did vary by sex and this relationship should be further investigated.     

Functionally Active Toll-Like Receptor 3 on Human Primary and Metastatic Cancer Cells

Toll-like receptor 3 (TLR3) is a member of Toll-like receptors who recognize structurally conserved molecules derived from pathogens and trigger the immune response. To clarify if TLR3, is expressed in certain tumour cell lines and whether it is functional and what is the response of these cell lines to different concentrations of poly I:C treatment, we have screened SW480, SW620, FaDu and Detroit 562 cell lines using real-time PCR, flow cytometry and ELISA. We have shown that all these cell lines express TLR3 on mRNA and protein level but it is only functional in Detroit 562 cell line since only in these cells poly I:C treatment triggered the IL-6 secretion. In addition, poly I:C treatment inhibited cell growth and triggered up-regulation of IL-12p40, IL-8 and Il-1α in Detroit 562 cell line. By using annexin-V apoptosis detection kit, we have found that poly I:C triggers apoptosis in Detroit 562 cell line. We have found here that based on the results of TLR3 functionality there is a huge difference between FaDu and Detroit 562 cell lines which are of the same origin (pharynx) but FaDu is primary and Detroit 562 metastatic carcinoma. Our study also shows that Detroit 562 cell line could be a good model for cancer therapy research and development as it is responsive to TLR3 agonists which consequently drives it to apoptosis.     

Fibroblast Growth Factor Receptor 1 Overexpression Is Associated with Poor Survival in Patients with Resected Muscle Invasive Urothelial Carcinoma

PurposeTo examine the usefulness of various receptor tyrosine kinase expressions as prognostic markers and therapeutic targets in muscle invasive urothelial cancer (UC) patients.ResultsThere were 41 (41.8%), 44 (44.9%), and 14 (14.2%) patients who have over-expressed HER2, FGFR1, and FGFR3, respectively. In univariate analysis, significantly shorter median time to recurrence (TTR) (12.9 months vs. 49.0 months; p=0.008) and overall survival (OS) (22.3 months vs. 52.7 months; p=0.006) was found in patients with FGFR1 overexpression. By contrast, there was no difference in TTR or OS according to the HER2 and FGFR3 expression status. FGFR1 remained as a significant prognostic factor for OS with hazard ratio of 2.23 (95% confidence interval: 1.27–3.90, p=0.006) in multivariate analysis.ConclusionOur result showed that FGFR1 expression, but not FGFR3, is an adverse prognostic factor in muscle invasive UC patients after radical cystectomy. FGFR1 might be feasible for prognosis prediction and a potential therapeutic target after thorough validation in muscle invasive UC.     

The Role of Toll-Like Receptor Signaling in Human Immunodeficiencies

Through the last decade, clinical immunology has witnessed a considerable progress in understanding the role of the innate immunity in human host defense, with Toll-like receptors (TLRs) being the most extensively innate immune receptors investigated. Growing literature documents the relevance of TLR signaling pathways to human disease, revealing a small, but expanding, group of new monogenic primary immunodeficiencies, in patients with various infectious diseases, previously considered as of unexplained “idiopathic” origin. Herein, we review these recently described deficiencies. Autosomal recessive IRAK-4 and myeloid differentiation factor 88 deficiencies were reported in 2003 and 2008, respectively, conferring predisposition to pyogenic bacterial infections, and autosomal recessive UNC93B1 and autosomal dominant TLR3 deficiencies were reported in 2006 and 2007, respectively, conferring predisposition to herpes simplex encephalitis. Furthermore, we highlight the published data associating TLR polymorphism with an altered susceptibility to infectious diseases.