Effect of activation of Toll-like receptor signaling pathway of peripheral blood mononuclear cell in recombinant hepatitis B surface antigen immune response / 中华流行病学杂志

Objective: To explore the effect and mechanism of activation of peripheral blood mononuclear cell (PBMC) Toll-like receptor (TLR3) signaling pathway in recombinant HBsAg (rHBsAg) immune response. Methods: White blood cells were collected from peripheral blood of 13 healthy donors in the preparation of blood products. PBMC was isolated and treated with Poly I:C (Poly I:C group) and PBS (control group) respectively. 48 h later, some cells were collected and the expressions of TLR3 signaling pathway proteins were detected by flow cytometry. After activating (Poly I:C group)/inactivating (control group) TLR3 signaling pathway, rHBsAg was given to both groups for 72 h, and the proportions of DC, T, B cells and their subsets in PBMC were detected by flow cytometry. Paired t-test, paired samples wilcoxon signed-rank test and canonical correlation analyses were used for statistical analysis. Results: The percentage of TLR3 protein-positive cells (19.21%) and protein expression (8 983.95), NF-κB protein expression (26 193.13), the percentage of pNF-κB protein-positive cells (13.73%) and its proportion in NF-κB (16.03%), and the percentage of pIRF3 protein-positive cells (12.64%) and its proportion in IRF3 (21.80%) in Poly I:C group were higher than those in control group (11.54%, 8 086.00, 22 340.66, 8.72%, 9.71%, 9.57%, 19.12%) (P<0.05), and the percentage of TRIF protein-positive cells (89.75%) and protein expression (304 219.54) were higher in Poly I:C group than in the control group (89.64%, 288 149.72) (P>0.05). After PBMC stimulation by rHBsAg, the proportions of mDC (2.90%), pDC (1.80%), B cell (5.31%) and plasma cell (67.71%) in Poly I:C group were significantly higher than those in the control group (1.83%, 0.81%, 4.23%, 58.82%) (P<0.05). Results of canonical correlation analysis showed that the expression of TLR3 protein was positively correlated with the proportions of plasma cells, the expression of pIRF3 protein was positively correlated with the proportions of plasma cells and mDC, and the percentage of pNF-κB protein-positive cells and the percentage of pIRF3 protein-positive cells were positively correlated with the proportion of CD4+T cells. Conclusions: Poly I:C can activate TLR3/TRIF/NF-κB and TLR3/TRIF/IRF3 signaling pathway, promote the function of downstream signaling molecules, and then promote the maturation of DC, induce the immune responses of CD4+T cell, and promote the maturation and activation of B cells and the immune response of rHBsAg.

Caracterização imunológica da formulação vacinal baseada em VLP (Virus Like Particle) da Proteína Circumsporozoíta de Plasmodium vivax / Immunological characterization of the VLP (Virus Like Particle) vaccine formulation fused to Circumsporozoite Protein of Plasmodium vivax

O Plasmodium vivax é a espécie mais comum de parasita causador da malária humana encontrada fora da África, com maior endemicidade na Ásia, América Central e do Sul e Oceania. Embora o Plasmodium falciparum cause a maioria do número de mortes, o P. vivax pode levar à malária grave e resultar em morbimortalidade significativa. O desenvolvimento de uma vacina protetora será um passo importante para a eliminação da malária. Recentemente, uma formulação contendo as três variantes alélicas da proteína circumsporozoíta de P. vivax (PvCSP - All epitopes) induziu proteção parcial em camundongos após desafio com esporozoíto híbrido Plasmodium berghei (Pb), no qual as repetições centrais do PbCSP foram substituídas por repetições PvCSP-VK210 (esporozoítos Pb/Pv). No presente estudo, a proteína quimérica PvCSP contendo as variantes alélicas (VK210, VK247 e P. vivax-like) fusionadas com a proteína de nucleocapsídeo do vírus da caxumba (formando partículas semelhantes a nucleocapsídeos ou do inglês, NLP - Núcleo Like Particles) na ausência (NLP-CSPR) ou na presença do domínio C-terminal (CT) conservado da PvCSP (NLP-CSPCT). Para a realização do estudo selecionamos os adjuvantes Poly (I:C), um RNA sintético de dupla fita, agonista do receptor Toll do tipo 3 (TLR3) ou o adjuvante Montanide ISA 720, uma emulação óleo em agua. Para obter uma forte resposta imune, a levedura Pichia pastoris foi usada para expressar as proteínas recombinantes na forma de NLPs. Camundongos foram imunizados com cada uma das proteínas recombinantes em combinação com os adjuvantes citados. Embora ambas as NLPs tenham sido capazes de gerar uma forte resposta imune, com altos níveis de títulos e longevidade, apenas a formulação contendo a proteína NLP-CSPCT na presença do adjuvante Poly (I:C) foi selecionada para ser explorada em experimentos futuros. Esta proteína em combinação com o adjuvante Poly (I:C) induziu alta frequência de células secretoras de anticorpos específicas para o antígeno homólogo nos dias 5 e 30, no baço e na medula óssea, respectivamente. Altos títulos de IgG contra as 3 variantes de PvCSP foram detectados nos soros. Posteriormente camundongos imunizados com NLP-CSPCT foram desafiados com esporozoítos Pb/Pv e a parasitemia no 5º dia demonstrou proteção estéril em 30% dos camundongos desafiados. Portanto, a formulação vacinal gerada neste estudo tem potencial para ser explorada no desenvolvimento de uma vacina universal contra a malária causada por P. vivax

Plasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP--All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and P. vivax-like) were fused with the mumps virus nucleocapsid protein (assembling into nucleo like particles - NLP) in the absence (NLP-CSPR) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSPCT). To carry out the study, we selected the adjuvants Poly (I:C), a synthetic double-stranded RNA, Toll-like receptor 3 (TLR3) agonist or Montanide ISA 720 adjuvant, an oil-water emulation. To elicit stronger immune response, Pichia pastoris yeast was used to produce the NLPs. Mice were immunized with each recombinant protein in combination with above. Although both NLPs were able to generate stronger immune response, with high antibodies titer levels and longevity, formulation containing NLP-CSPCT in the presence of Poly (I:C) was selected to be explored in future experiments. NLP-CSPCT with Poly (I:C) adjuvant presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, immunized mice with NLP-CSPCT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria.

Association of TLR3 single nucleotide polymorphisms with susceptibility to HTLV-1 infection in Iranian asymptomatic blood donors

Abstract INTRODUCTION: The human T-lymphotropic virus type 1 (HTLV-1) has a single-stranded RNA genome and expresses specific proteins that have oncogenic potential. Approximately 15 to 20 million people worldwide have been infected by this virus. Changes in protein or gene expression are the effects of single nucleotide polymorphisms (SNPs) within the Toll-like receptor 3 (TLR3) gene. The function and efficacy of signal transduction also lead to modified immune responses. The present study aimed to investigate the association of SNPs within TLR3 (rs3775291 and rs3775296) with susceptibility to HTLV-1 infection in Iranian asymptomatic blood donors. METHODS: This study was performed on 100 HTLV-1-infected asymptomatic blood donors and 118 healthy blood donors. Genomic DNA from all participants was purified and then amplified using specific PCR primers. SNPs within TLR3 were evaluated using the restriction fragmentation length polymorphism technique, and the results were analyzed using SPSS software (version 22). RESULTS: The frequencies of the TLR3 (rs3775296) CC, CA, AA genotypes were 70%, 24%, and 6% in the patient group, and 50.8%, 44.9%, and 4.2% in the control group, respectively. There was a significant difference in the frequency distribution of TLR3 (rs3775296) genotypes and alleles, but not in the frequency distribution of TLR3 (rs3775291) genotypes between the patient and control groups. CONCLUSIONS: The TLR3 SNP rs3775296 was significantly associated with HTLV-1 infection and may be a protective factor against this viral infection.

Evaluation of different total Leishmania amazonensis antigens for the development of a first-generation vaccine formulated with a Toll-like receptor-3 agonist to prevent cutaneous leishmaniasis

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Evaluation of variants in IL6R, TLR3, and DC-SIGN genes associated with dengue in a sampled Colombian population / Evaluación de las variantes en los genes IL6R, TLR3 y DC-SIGN asociadas con dengue en una muestra de población colombiana

Abstract Introduction: Host genetics is recognized as an influential factor for the development of dengue disease. Objective: This study evaluated the association of dengue with the polymorphisms rs8192284 for gene IL6R, rs3775290 for TLR3, and rs7248637 for DC-SIGN. Materials and methods: Of the 292 surveyed subjects, 191 were confirmed for dengue fever and the remaining 101 were included as controls. The genotypes were resolved using polymerase chain reaction and restriction fragment length polymorphism (PCR- RFLP). In an attempt to determine the risk (Odds Ratio) of suffering dengue fever, data were analyzed using chi-square for alleles and logistic regression for both genotypes and allelic combinations. Confidence intervals were set to 95% for all tests regardless of the adjustment by either self-identification or ancestry. Results: For Afro-Colombians, the allele rs8192284 C offered protection against dengue [OR=0.425,(0.204-0.887), p=0.020]. The alleles rs7248637 A and rs3775290 A posed, respectively, an increased risk of dengue for Afro-Colombians [OR=2.389, (1.170-4.879), p=0.015] and Mestizos [OR=2.329, (1.283-4.226), p=0.005]. The reproducibility for rs8192284 C/C [OR=2.45, (1.05-5.76), p=0.013] remained after adjustment by Amerindian ancestry [OR=2.52, (1.04-6.09), p=0.013]. The reproducibility for rs3775290 A/A [OR=2.48, (1.09-5.65), p=0.033] remained after adjustment by European [OR=2.34, (1.02-5.35), p=0.048], Amerindian [OR=2.49, (1.09-5.66), p=0.035], and African ancestry [OR=2.37, (1.04-5.41), p=0.046]. Finally, the association of dengue fever with the allelic combination CAG [OR=2.07, (1.06-4.05), p=0.033] remained after adjustment by Amerindian ancestry [OR=2.16, (1.09-4.28), p=0.028]. Conclusions: Polymorphisms rs8192284 for IL6R, rs3775290 for TLR3, and rs7248637 for DC-SIGN were associated with the susceptibility to suffer dengue fever in the sampled Colombian population.

Resumen Introducción. La genética del huésped se reconoce como un factor que influye en el desarrollo del dengue. Objetivo. Este estudio evaluó la asociación del dengue con los polimorfismos rs8192284 del gen IL6R, rs3775290 del TLR3 y rs7248637 del DC-SIGN. Materiales y métodos. De los 292 sujetos encuestados, en 191 se confirmó la presencia de fiebre por dengue y los restantes 101 se incluyeron como controles. Los genotipos se resolvieron mediante reacción en cadena de la polimerasa y polimorfismos en la longitud de los fragmentos de restricción (PCR-RFLP). En un intento por determinar el riesgo de sufrir dengue, los datos se analizaron mediante la prueba de ji al cuadrado para los alelos y la regresión logística para los genotipos y las combinaciones alélicas. Los intervalos de confianza se calcularon a 95 % para todas las pruebas independientemente ajustadas por autoidentificación o componente genético ancestral. Resultados. En los afrocolombianos, el alelo C rs8192284 ofreció protección contra el dengue (OR=0,425; 0,204-0,887, p=0,020). Los alelos A rs7248637 y Ars3775290 plantearon un mayor riesgo de dengue para los afrocolombianos (OR=2,389; 1,170- 4,879; p=0,015) y los mestizos (OR=2,329; 1,283-4,226: p=0,005), respectivamente. La reproducibilidad para rs8192284 C/C (OR=2,45; 1,05-5,76; p=0,013) permaneció después del ajuste por el componente genético ancestral amerindio (OR=2,52; 1,04- 6,09; p=0,013). La reproducibilidad del rs3775290 A/A (OR=2,48; 1,09-5,65; p=0,033) permaneció después del ajuste por el componente europeo (OR=2,34; 1,02-5,35; p=0,048), el amerindio (OR=2,49; 1,09- 5,66; p=0,035), y el africano (OR=2,37; 1,04- 5,41; p=0,046). Por último, la asociación del dengue con la combinación alélica CAG (OR=2,07; 1,06-4,05; p=0,033) permaneció después del ajuste por el componente genético amerindio (OR=2,16; 1,09-4,28;p=0,028). Conclusión. Los polimorfismos rs8192284 en IL6R, rs3775290 en TLR3 y rs7248637 en DC-SIGN, se asociaron con la propensión a sufrir dengue en una muestra de población colombiana.

Influência de TLR3 em modelo de Paracoccidioidomicose experimental / TLR3 influence in experimental PCM model

Os receptores do tipo Toll compreendem a família de receptores de reconhecimento de padrões melhor caracterizados, que podem ativar diferentes respostas imunes, dependendo de quais receptores e conjuntos de adaptadores são utilizados. Os TLRs, como TLR2, TLR4 e TLR9, e sua sinalização foram implicados no reconhecimento de P. brasiliensis e na regulação da resposta imune, no entanto, o papel do TLR3 ainda não está claro. Assim, a compreensão da função endossomal do TLR3 na PCM experimental é crucial. Utilizamos modelos in vitro e in vivo de infecção por P. brasiliensis, camundongos C57Bl/6 e TLR3-/-, para avaliar a contribuição da TLR3 no desenvolvimento da infecção. Mostramos que ausência de TLR3 leva o aumento de óxido nítrico e a capacidade fagocítica por macrófagos nas primeiras 4 horas de interação com leveduras P. brasiliensis. Mostramos ainda que os camundongos TLR3-/- desempenham papel protetor após 30 dias de infecção intratraqueal com P. brasiliensis, mostrando diminuição do aumento de CFU, perfil de resposta Th1 e Th17, bem como aumento de células citotóxicas T CD8+ produtoras de IFN-γ e IL-17. As células citotóxicas T CD8+ mostraram ser essenciais para o controle da infecção nos camundongos TLR3-/-, uma vez que a depleção dessas células levou a progressão da doença. Em estágios iniciais, 3 e 5 dias de infecção, observamos aumento do recrutamento de neutrófilos para o pulmão. Estudos recentes indicam que o TLR3 é um receptor importante para a resposta imune na micose e sua ausência favorece a infecção por fungos. Em contraste, nossos resultados mostram que, no caso do PCM, o TLR3 é prejudicial ao hospedeiro, sugerindo que a ativação do TLR3 pode ser um possível mecanismo de escape de P. brasiliensis

Toll-like receptors comprise the best-characterized pattern-recognition receptor family that can activate different immune responses, depending on which receptor and adaptor set are utilized. TLRs, such as TLR2, TLR4 and TLR9, and their signaling have been implicated in the recognition of P. brasiliensis and regulation of the immune response, however, the role of TLR3 remains unclear. Thus, understanding the endosomal function of TLR3 in experimental PCM is crucial. We used in vitro and in vivo models of infection by P. brasiliensis, C57Bl/6 and TLR3-/- mice, to assess the contribution of TLR3 on development of infection. We show that absence of TLR3 leads to increased nitric oxide and phagocytic capacity by macrophages in the first 4 hours of interaction with yeasts P. brasiliensis. We also showed that TLR3-/- mice play a protective role after 30 days of intratracheal infection with P. brasiliensis, showing a decrease in the CFU increase, Th1 and Th17 response profile, as well as an increase in cytotoxic CD8+ cells producing IFN-γ and IL-17. The cytotoxic T CD8+ cells were shown to be essential for the control of infection in TLR3-/- mice, since the depletion of these cells led to the progression of the disease. In the initial stages, 3 and 5 days of infection, we observed increased recruitment of neutrophils to the lung. Recent studies indicate that TLR3 is an important receptor for the immune response in mycosis and its absence favors fungal infection. In contrast, our results show that in the case of PCM, TLR3 is detrimental to the host, suggesting that TLR3 activation may be a possible escape mechanism of P. brasiliensis

TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling

PURPOSE: Tripartite-motif-containing protein 56 (TRIM56) has been found to exhibit a broad antiviral activity, depending upon E3 ligase activity. Here, we attempted to evaluate the function of TRIM56 in multiple myeloma (MM) and its underlying molecular basis. MATERIALS AND METHODS: TRIM56 expression at the mRNA and protein level was measured by qRT PCR and western blot analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry analysis was performed to investigate the effect of TRIM56 on MM cell proliferation and apoptosis. The concentrations of interferon (IFN)-β, interleukin (IL)-6, and tumor necrosis factor-α in MM cell culture supernatants were detected with respective commercial ELISA kits. Western blot was employed to determine the effect of TRIM56 on toll-like receptor 3 (TLR3)/toll-IL-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) signaling pathway. RESULTS: TRIM56 expression was prominently decreased in MM cells. Poly (dA:dT)-induced TRIM56 overexpression in U266 cells suppressed proliferation, induced apoptosis, and enhanced inflammatory cytokine production, while TRIM56 knockdown improved growth, diminished apoptosis, and inhibited inflammatory cytokine secretion in RPMI8226 cells. Moreover, TRIM56 knockdown blocked TLR3 signaling pathway. Furthermore, poly (I:C), a TLR3 agonist, markedly abolished TRIM56 depletion-induced increase of proliferation, decrease of apoptosis, and reduction of inflammatory factor in MM cells. CONCLUSION: TRIM56 may act as a tumor suppressor in MM through activation of TLR3/TRIF signaling pathway, contributing to a better understanding of the molecular mechanism of TRIM56 involvement in MM pathogenesis and providing a promising therapy strategy for patients with MM.

Sex hormones alter the response of Toll-like receptor 3 to its specific ligand in fallopian tube epithelial cells / 대한생식의학회지

OBJECTIVE: The fallopian tubes play a critical role in the early events of fertilization. The rapid innate immune defense is an important part of the fallopian tubes. Toll-like receptor 3 (TLR3), as a part of the innate immune system, plays an important role in detecting viral infections. In this basic and experimental study, the effect of sex hormones on the function of TLR3 in the OE-E6/E7 cell line was investigated. METHODS: The functionality of TLR3 in this cell line was evaluated by cytokine measurements (interleukin [IL]-6 and IL-1b) and the effects of sex hormones on TLR3 were tested by an enzyme-linked immunosorbent assay kit. Additionally, TLR3 small interfering RNA (siRNA) and a TLR3 function-blocking antibody were used to confirm our findings. RESULTS: The production of IL-6 significantly increased in the presence of polyinosinic-polycytidylic acid (poly(I:C)) as the TLR3 ligand. Using a TLR3-siRNA-ransfected OE-E6/E7 cell line and function-blocking antibody confirmed that cytokine production was due to TLR3. In addition, 17-β estradiol and progesterone suppressed the production of IL-6 in the presence and absence of poly(I:C). CONCLUSION: These results imply that sex hormones exerted a suppressive effect on the function of TLR3 in the fallopian tube cell line when different concentrations of sex hormones were present. The current results also suggest that estrogen receptor beta and nuclear progesterone receptor B are likely to mediate the hormonal regulation of TLR3, as these two receptors are the main estrogen and progesterone receptors in OE-E6/E7 cell line.

Association of TLR3-1377C/T gene polymorphisms and expression with susceptibility to enterovirus 71 encephalitis in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the association of gene polymorphisms of Toll-like receptor 3 (TLR3)-1377C/T and expression of TLR3 with the susceptibility to enterovirus 71 (EV71) encephalitis in children.</p><p><b>METHODS</b>A total of 187 children with EV71 infection (59 children in the encephalitis group and 128 in the non-encephalitis group) and 232 children who underwent physical examination were enrolled in the case-control study. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the TLR3-1377C/T gene polymorphisms. ELISA was used to measure the serum level of TLR3.</p><p><b>RESULTS</b>There were no significant differences in the genotype and allele frequencies of TLR3-1377C/T between the non-encephalitis group and the encephalitis group. Compared with the control group, the encephalitis group and the non-encephalitis group had significant increases in the serum level of TLR3 (P<0.05), and the non-encephalitis group had the highest level (P<0.05). The encephalitis group had a significantly higher EV71 viral load than the non-encephalitis group (P<0.01). The children aged <1 year or ≥1 year in the encephalitis group and the non-encephalitis group had significant increases in the serum level of TLR3 compared with their counterparts in the control group (P<0.05), and the children aged <1 year or ≥1 year in the non-encephalitis group had a significantly higher serum level of TLR3 than those in the encephalitis group (P<0.05). In the encephalitis group, the children aged ≥1 year had a significantly higher TLR3 concentration than those aged <1 year (P<0.05), and there were no significant differences in the TLR3 concentration between the children aged ≥1 year and <1 year in the non-encephalitis group and the control group. In the encephalitis group, the proportion of children aged <1 year was significantly higher than those aged ≥1 year (P<0.05).</p><p><b>CONCLUSIONS</b>The TLR3-1377C/T gene polymorphisms are not significantly associated with the development of EV71 encephalitis. Low expression of TLR3 might weaken the inhibitory effect on virus replication and promote the development of EV71 encephalitis. The deficiency in the expression of TLR3 in serum after EV71 infection might be an important factor for the development of encephalitis in infants.</p>

Studying the Effect of Downregulating Autophagy-Related Gene LC3 on TLR3 Apoptotic Pathway Mediated by dsRNA in Hepatocellular Carcinoma Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The purpose of this study is to examine the role of the double-stranded RNA (dsRNA) activated Toll–interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF) signal pathway in triggering apoptosis in hepatocellular carcinoma (HCC) cells. MATERIALS AND METHODS: First, siRNA targeted autophagy–related gene LC3 (pU6H1-LC3 siRNA and siLC3) and a dsRNA used as a Toll-like receptor 3 (TLR3) ligand was constructed and synthesized, respectively. Then, a human HCC cell line was transfected with dsRNA, siLC3, and cotransfected with siLC3 and dsRNA (siLC3+dsRNA), respectively. Finally, quantification real-time polymerase chain reaction, western blotting, and immunofluorescence staining were used in the HCC line (SMMC7721), and MTT assay, flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, and transmission electron microscopy were used in an HCC xenograft model of nude mice. Human umbilical vein endothelial cell tube forming assay, color Doppler ultrasonographic flow image examination, and CD34-positive microvessel density were used in vitro and in vivo. RESULTS: Compared with untreated cells, the protein and mRNA expression of TLR3 and TRIF was up-regulated, in order, siLC3+dsRNA, dsRNA, and siLC3. Expression of LC3 was obviously down-regulated and the autophagosomes were significantly decreased in siLC3+dsRNA and siLC3, whereas in dsRNA (p < 0.05). LC3 and TRIF colocation was observed in HepG2 cells. Decreased cell viability, increased apoptosis, decrease in xenograft tumor volume, and angiogenesis potential were also observed in order (p < 0.05). CONCLUSION: Suppression of intracellular autophagy resulted in decreased degradation of TRIF protein, which can promote triggering of apoptosis by the TLR3-TRIF pathway. dsRNA and siLC3 could play anticancer roles in coordination.

Association of TLR3 gene polymorphism with IgG subclass deficiency and the severity in patients with aspirin-intolerant asthma

PURPOSE: Toll-like receptor 3 (TLR3) recognizes to viral double-stranded RNA and is involved in antiviral defenses. A probable role of TLR3 gene variants in the pathogenesis of aspirin-intolerant asthma (AIA) has been suggested. AIA patients present more frequent asthma exacerbations in which respiratory viral infections could be an exacerbating factor. IgG subclass deficiency was commonly present with bronchial asthma. Based on previous findings, we investigated whether TLR3 variants could affect IgG3 subclass deficiency in AIA. METHODS: We enrolled 279 AIA patients, 403 aspirin-tolerant asthma (ATA) patients, and 315 normal healthy controls (NC) in this study. TLR3 polymorphism at the promoter region -299698G>T was genotyped. The serum levels of IgG subclasses were determined by the single radial immunodiffusion method. Expressions of IgG3 and TLR3 on Epstein-Barr virus transformed-B cells isolated from asthmatic patients were evaluated by flow cytometry to investigate B-cell functions. RESULTS: The TLR3 -299698 T allele was significantly associated with severity and IgG3 deficiency in the AIA group (P=0.044 and P=0.010, respectively), but not in the ATA group. IgG3 expression on B cells from asthmatics with IgG3 deficiency was significantly lower compared to those without (P=0.025). There was a positive correlation between IgG3 expression levels on B cells and serum IgG3 levels (r 2=0.434, P=0.002). CONCLUSION: These results suggest that the TLR3 -299698G>T polymorphism may be associated with IgG3 subclass deficiency and severity in AIA.

Heparin-treated dendritic cells promote Th0 to Th1 differentiation via the Toll-like receptor 3 in peripheral blood monocytes of patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the mechanisms underlying the ability ofheparin-treated dendritic cells (DCs) to promote Th0 to Th1 differentiation in chronic hepatitis B (CHB).</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMCs) were isolated from CHB patients and cultured in RPMI-1640 with recombinant GM-CSF and IL-4 with or without heparin to obtain DCs for study. The levels of Toll-like receptors (TLRs) on the DCs were measured using FACS and qPCR techniques.DC subsets with high expression of TLRs were selected for analysis of functional changes by treatment with the corresponding TLR-siRNA. The CD4+ T cell subpopulation was purified from peripheral blood by Dynal immunomagnetic beads, and then the production of IL-12 by DCs in the presence of poly(I:C) or R848 and ofIFN and IL-4 by Th cells co-cultured with DCs was evaluated by ELISA. The t-test was used for statistical analysis.</p><p><b>RESULTS</b>TLR3 expression, and not expression of TLR 7 or TLR8,was significantly increased in heparin-treated DCs as compared to levels detected in the DCs without heparin treatment (t =2.849,P less than 0.05;t =3.027,P less than 0.05). The level of IL-12 produced by heparin-treated DCs stimulated with poly(I:C) was obviously higher than that produced by DCs without heparin treatment and stimulated with poly(I: C) (t =8.68,P less than 0.01) or with R848 (t =19.01,P less than 0.01). However, the IL-12 production by TLR3-siRNA transfected-DCs was significantly reduced (t =31.49, P less than 0.01).When Th cells from allogenic patients with CHB were co-cultured with the TLR3-siRNA transfectedDCs, the frequency ofCD4+ IFN+ cells was significantly reduced (1.64+/-0.57% vs.6.31+/-0.88%,P less than 0.01),as was the capability of Thl to generate IFNg (t =20.83,Pless than 0.01).</p><p><b>CONCLUSION</b>Heparin may have up-regulated the TLR3 expression level of DCs, and sequentially promoted Th0 to Th1 differentiation.</p>

Clinical characteristics, prognosis and genetic susceptibility of herpes simplex encephalitis in children / 中华儿科杂志

<p><b>OBJECTIVE</b>To summarize the clinical characteristics and long-term prognosis of herpes simplex virus encephalitis (HSE) in childhood and to analyze genotype of UNC93B1 and TLR3.</p><p><b>METHOD</b>Data of a total of 30 HSE patients admitted to Beijing Children's Hospital from January 2008 to September 2013 were retrospectively analyzed, the data included clinical manifestations, physical sign, auxiliary examination, therapy and long-term clinical prognosis. The family history obtained during follow-up visit was also analyzed for genetic predisposition. With parents' agreement, the blood specimens of patients were collected in EDTA anticoagulant tubes, the first 2 genetic etiologies UNC93B1 and TLR3 were sequenced, and the genetic susceptibility to HSE in childhood was summarized.</p><p><b>RESULT</b>(1) All the 30 patients (100%) had fever, 28 (93%) had seizure, 25 (83%) had altered state of consciousness, only 11 (37%) had personality changes, and in 8 (73%) appeared at or after 2 weeks of onset . (2) During the long-term follow up, 2 (7%) patients died after discharge, 23 patients (82%) had neurological sequelae, 13 patients (57%) had moderate, severe disability and vegetative state. (3) After sequencing of UNC93B1, and TLR3, one patient was found homozygous for a single-nucleotide substitution at position C.414C>G in exon 4 of UNC93B1 which affected the expression of UNC93B1, and may block or decrease the production of interferon. (4) Six single nucleotide polymorphisms (SNPs) were found in this study, their genotype frequency and gene frequency of Chinese were respectively searched in Genomes Project in NCBI and defined 1 000 genomes group. The genotype frequency of UNC93B1 rs7149 between 1 000 genomes group and HSE group was significantly different (χ² = 55.37, P<0.05). The frequency of CC type and C type was higher in HSE group, both of them had significant difference (χ² = 93.90, P<0.05, OR=61.563; χ² = 134.40, P<0.05, OR=12.491).</p><p><b>CONCLUSION</b>HSE lacks specific clinical manifestations, the long-term prognosis is poor. One HSE patient carrying a heterozygous mutation in UNC93B1 which may lead to the susceptibility to HSE and had harmful effect on long-term prognosis. The SNP UNC93B1 rs7149 may also have relationship with susceptibility to HSE and the children carrying CC genotype or C gene in this gene site maybe more susceptible to HSE.</p>

TLR3 gene polymorphisms in cancer: a systematic review and meta-analysis / 癌症

<p><b>INTRODUCTION</b>Recent studies examining the association of Toll-like receptor 3 (TLR3) gene polymorphisms with the risk of developing various types of cancer have reported conflicting results. Clarifying this association could advance our knowledge of the influence of TLR3 single nucleotide polymorphisms (SNPs) on cancer risk.</p><p><b>METHODS</b>We systematically reviewed studies that focused on a collection of 12 SNPs located in the TLR3 gene and the details by which these SNPs influenced cancer risk. Additionally, 14 case-control studies comprising a total of 7997 cases of cancer and 8699 controls were included in a meta-analysis of 4 highly studied SNPs (rs3775290, rs3775291, rs3775292, and rs5743312).</p><p><b>RESULTS</b>The variant TLR3 genotype rs5743312 (C9948T, intron 3, C>T) was significantly associated with an increased cancer risk as compared with the wild-type allele (odds ratio [OR]=1.11, 95% confidence interval [CI]=1.00-1.24, P=0.047). No such association was observed with other TLR3 SNPs. In the stratified analysis, the rs3775290 (C13766T, C>T) variant genotype was found to be significantly associated with an increased cancer risk in Asian populations. Additionally, the rs3775291 (G13909A, G>A) variant genotype was significantly associated with an increased cancer risk in Asians, subgroup with hospital-based controls, and subgroup with a small sample size.</p><p><b>CONCLUSION</b>After data integration, our findings suggest that the TLR3 rs5743312 polymorphism may contribute to an increased cancer risk.</p>

Regulation of Jinxin Oral Liquid for the expression of negative regulatory factor of TLR3 signaling pathway SOCS1 in RSV infected BALB/c mice / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the regulation trend of Jinxin Oral Liquid (JXOL) on the expression of negative regulatory factor of TLR3 signaling pathway SOCS1 in the lung tissue of RSV infected BALB/c mice at different time points.</p><p><b>METHODS</b>Totally 75 BALB/c mice were randomly divided into 5 groups, i.e., the normal control group, the model group, the ribavirin group, the high dose JXOL group, and the equivalent dose JXOL group, 15 in each group. Each group had 3 intervention ways (I, II, and III) with 5 mice treated in each group. BALB/c mice were nasally infected with respiratory syncytial virus (RSV), and treated by different intervention ways. After intervention, mice were killed and their lung tissues were sampled, mRNA expression levels of RSV-M, SOCS1, and IFN-β were detected by Real time PCR. The expression of SOCSl at the protein level was detected by Western blot.</p><p><b>RESULTS</b>Compared with the normal control group, the mRNA expression level of SOCS1 and IFN-β, and the protein expression level of SOCS1 increased significantly in the model group intervened by intervention I and II (all P < 0.01), but the mRNA expression level of IFN-β decreased significantly in model group intervened by intervention III (P < 0.01). Compared with the model group, the mRNA expression level of RSV-M all significantly decreased in the high dose JXOL group and the equivalent dose JXOL group intervened by 3 intervention ways (all P < 0.01). The mRNA expression level of SOCS1 significantly decreased in the high dose JXOL group intervened by intervention I and III and the equivalent dose JXOL group intervened by 3 intervention ways (all P < 0.01). The mRNA expression level of IFN-β significantly decreased in the high dose JXOL group intervened by intervention I and II and the equivalent dose JXOL group intervened by intervention I (all P < 0.01), while it significantly increased in the high dose JXOL group intervened by intervention III and the equivalent dose JXOL group intervened by intervention III (all P < 0.01). The protein expression level of SOCS1 significantly decreased in the high dose JXOL group intervened by intervention I and the equivalent dose JXOL group intervened by 3 intervention ways (all P < 0.01), while it significantly increased in the high dose JXOL group intervened by intervention III (all P < 0.01). Compared with the high dose JXOL group, the mRNA expression level of RSV-M decreased significantly in the equivalent dose JXOL group intervened by intervention I and II (P < 0.01). The mRNA expression level of SOCS1 and IFN-β decreased significantly in the equivalent dose JXOL group intervened by intervention I (P < 0.01), but the mRNA expression level of IFN-β increased significantly in the equivalent dose JXOL group intervened by intervention II and III (all P < 0.01). The protein expression level of SOCS1 decreased significantly in the equivalent dose JXOL group intervened by 3 intervention ways (all P < 0.01).</p><p><b>CONCLUSIONS</b>JXOL could inhibit the expression of SOCS1 in the lung tissue of RSV infected BALB/c mice at different time points. Its regulatory effect might be associated with promoting the expression of interferon type I and further fighting against RSV.</p>

Polyinosine-polycytidylic acid promotes excessive iodine intake induced thyroiditis in non-obese diabetic mice via Toll-like receptor 3 mediated inflammation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Excessive iodine intake and viral infection are recognized as both critical factors associated with autoimmune thyroid diseases. Toll-like receptors (TLRs) have been reported to play an important role in autoimmune and inflammatory disorders. In this study, we aimed to clarify the possible mechanism of TLR3 involved in polyinosine-polycytidylic acid (poly(I:C)) promoting excessive iodine intake induced thyroiditis in non-obese diabetic (NOD) mice.</p><p><b>METHODS</b>Both NOD and BALB/c mice were randomly assigned to four groups: control group (n = 5), high iodine intake (HI) group (n = 7), poly(I:C) group (n = 7) and combination of excessive iodine and poly(I:C) injection (HIP) group (n = 7). After 8 weeks, mice were weighed and blood samples were collected. All the mice were sacrificed before dissection of spleen and thyroid gland. Then, thyroid histology, thyroid secreted hormone, expression of CD3(+) cells and TLR3 as well as inflammatory mRNA level were evaluated.</p><p><b>RESULTS</b>Both NOD and BALB/c mice from HI and HIP group represented goiter and increasing thyroid relative weight. Thyroid histology evidence indicated that only HIP group of NOD mice showed severe thyroiditis with lymphocytes infiltration in majority of thyroid tissue, severe damage of follicles and general fibrosis. Immunofluorescence staining results displayed a large number of CD3(+) cells in HIP NOD mice. Real-time polymerase chain reaction (PCR) results suggested interferon (IFN)-α increased over 30 folds and IFN-γ expression was doubled compared with control group, but interleukin (IL)-4 remained unchanged in HIP group of NOD mice thyroid. Meanwhile, over one third decrease of blood total thyroxine (TT4) and increased thyroid-stimulating hormone (TSH) was observed in HIP group of NOD mice. Only HIP group of NOD mice represented significantly elevation of TLR3 expression.</p><p><b>CONCLUSION</b>Poly(I:C) enhanced excessive dietary iodine induced thyroiditis in NOD mice through increasing TLR3 mediated inflammation.</p>

Dynamic changes in PD-1, TLR3, and TLR4 surface expression on peripheral blood mononuclear cells in chronic hepatitis C patients undergoing peg-IFNalpha-2a plus ribavirin combination therapy / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic changes in expression of programmed death (PD)-1, Toll-like receptor (TLR)3, and TLR4 on the surface of peripheral blood mononuclear cells (PBMCs) in patients with chronic hepatitis C (CHC) that occur in response to pegylated-interferon alpha-2a (peg-IFNalpha-2a) plus ribavirin (RBV) combination therapy, and to analyze the relation to achievement of sustained virological response (SVR). METHODS Twenty-three CHC patients and 10 healthy controls were enrolled in the study. All CHC patients underwent 48 weeks of combination therapy with peg-IFNalpha-2a (180 microg, subcutaneous injection, once weekly) plus RBV (15 microg/kg, oral, once daily). Total PBMCs were isolated from both groups (CHC patients at treatment week 0, 12, 24, and 48 and post-treatment week 24; controls at enrollment) and subjected to flow cytometric analysis of PD-1, TLR3, and TLR4 surface expression. In addition, serum levels of alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels were analyzed by enzymatic assay and the AmpliPrep/COBAS (Roche) nucleic acid amplification test, respectively. SVR was defined as undetectable levels of HCV RNA at post-treatment week 24. Intergroup differences were assessed by one-way ANOVA.</p><p><b>RESULTS</b>The expression ratios of PD-1, TLR4 and PD-1: TLR4 on PBMCs were significantly higher in CHC patients before therapy than in the healthy controls (45.20 +/- 7.12% vs. 16.82 +/- 4.13%, 58.45 +/- 15.13% vs. 21.09 +/- 2.89%, and 35.54 +/- 7.69% vs. 14.12 +/- 2.89%; all P < 0.05). In contrast, the expression ratios of TLR3 and PD-1:TLR3 were slightly, but not significantly, higher in CHC patients before therapy than in the healthy controls (P > 0.05). During the course of peg-IFNalpha-2a plus RBV combination therapy, the expression ratios of PD-1 and TLR4 on PBMCs showed a decreasing trend, while TLR3 expression showed an increasing trend. Furthermore, CHB patients who achieved SVR at post-treatment week 24 had a significantly different expression ratio of PD-1 and TLR3 than those who did not achieve SVR (P < 0.05).</p><p><b>CONCLUSION</b>Surface expression of PD-1, TLR4, and PD-1:TLR4 is up-regulated in the total PBMCs of CHC patients. Peg-IFNalpha-2a plus RBV treatment-induced suppression of HCV replication results in a significant reduction in PD-1 and TLR4 expression on the surface of PBMCs, but a remarkably elevated level of TLR3 expression. The dynamic change in PD-1 and TLR3 expression on PBMCs that occurs during antiviral therapy may be related to achievement of SVR.</p>

The antiviral role of Toll-like receptor 3 in human lung epithelial cells infected with respiratory syncytial virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>In order to understand the production mechanism of interferon and provide a scientific basis for preventionand clinical therapy, the expression changes of Toll-like receptor (TLR3) mRNA and the role of TLR3 in human lung epithelial cells (A549 cells) infected with respiratory syncytial virus (RSV) were investigated in this study.</p><p><b>METHODS</b>RSV infected A549 cells were treated with or without specific antibodies of TLR3 and collected at the selected timepoints after RSV infection (4, 8, 12, 16 and 24h). The expressions of TLR3, IFN-alpha, IFN-beta and RSV F mRNA were evaluated by RT-PCR.</p><p><b>RESULT</b>It was found that RSV infection could markedly up-regulate the mRNA expression of TLR3, IFN-alpha, IFN-beta and RSV F protein in a time-dependent manner as the 24h mRNA expressions of them were 4 times, 3 times, 3 times and 0.7 times more than the basic expression, respectively. Treatment of TLR3 specific antibodies, whereas, significantly down-regulated the activation of TLR3. The mRNA expression of IFN-alpha and IFN-1beta also decreased accordingly and that of IFN-beta reduced more obviously than IFN-alpha, but that of RSV F protein rose significantly.</p><p><b>CONCLUSION</b>Above data indicate that RSV infection could induce an apparent increase of antiviral genes of IFN-alpha and IFN-beta by activating TLR3 in human lung epithelial cells and the activated cells mediated Type I interferon is antiviral, which suggesting that TLR3 might play an important role in antiviral activity of RSV-infected human lung epithelial cells.</p>

Promoter region polymorphism & expression profile of toll like receptor-3 (TLR-3) gene in chronic hepatitis C virus (HCV) patients from India.

Background & objectives: Hepatitis C virus (HCV) induces an immune response of the host, manifested by the formation of anti-HCV antibodies mediated by adaptive and innate immunity. Toll-like receptors (TLRs) play a pivotal role in innate immunity system. This study was aimed to investigate the promoter region polymorphism and expression of TLR3 gene in patients with chronic HCV infection. Methods: Patients with chronic HCV infection (N=180) and an equal number of age-sex matched controls were included in the study. Patients positive for HCV-RNA were subjected to analysis of TLR3 polymorphism by direct sequencing of PCR products verified by comparing with the sequences reported in the National Centre for Biotechnology Information (NCBI) database (accession number: NT 022792). Expression of TLR3 gene was analyzed by semiquantitative RT-PCR using housekeeping β-actin gene as the internal control. Results: Polymorphisms at position -288G/A and -705A/G were identified. The results were significant in -705 allele (P=0.004) OR 2.79(1.46-5.42) and were associated with high risk of HCV infection. In silico sequence analysis showed the presence of ectropic viral integration site 1 encoded factor, in which G at -705 results in the loss of this site. The -7C/A polymorphism was not seen in our study cohort. The expression of TLR3 was upregulated in chronic HCV patients compared to healthy controls. Interpretation & conclusions: Polymorphism in the -705A/G allele at the promoter region of the TLR3 gene may predispose individual to HCV infection. However association of TLR3 expression with polymorphism of TLR3 promoter was not found.

Expressions of Toll-like receptors 3, 4, 7, and 9 in cervical lesions and their correlation with HPV16 infection in Uighur women / 癌症

Recent findings show that Toll-like receptors (TLRs) expressed in immune cells play a crucial role in the innate immune response and the subsequent induction of adaptive immune responses against microbial infection on tissue injury. Furthermore, expression of TLRs in cancer cells is associated with tumor proliferation and invasion. To explore the role of TLRs expression in cervical carcinogenesis in Uighur women, we detected the expressions of TLR3, TLR4, TLR7, and TLR9 in 25 normal cervical tissues, 64 cervical intraepithelial neoplasia (CIN) tissues, and 63 cervical squamous cell carcinoma (CSCC) tissues using immunohistochemical staining, as well as human papillomavirus type 16 (HPV16) infection using PCR. All samples used in this study were from Xinjiang Uighur women. We found the expression levels of TLR4, TLR7, and TLR9 were significantly higher in CIN and CSCC than in normal controls (P < 0.05). Up-regulation of TLR4 and TLR7 were correlated with tumor differentiation but not FIGO stage or lymph node metastasis (P > 0.05). Up-regulation of TLR9 was correlated with lymph node metastasis (P < 0.05) but not tumor differentiation or FIGO stage (P > 0.05). We also analyzed the correlation between the expressions of TLRs and HPV16 infection and found that the expressions of TLR4 and TLR9 significantly correlated with HPV16 infection in CIN (r = 7.434, P = 0.006; r = 7.123, P = 0.008) and CSCC (r = 6.423, P = 0.001; r = 8.478, P = 0.004), whereas the expression of TLR3 was not significantly different in any of the three groups and had no significant correlation with HPV16 infection. Our results suggest that high expression of TLR4, TLR7, and TLR9 may play important roles in the development and progression of CIN and CSCC in Uighur women, and the expressions of TLR4 and TLR9 can be up-regulated by HPV16 infection.