On the Impairment of Stress-Induced Changes in Triglyceride Levels via a Sub-Toxic Dose of Unmethylated Cytidine Phosphate Guanosine Oligodinucleotide (a Toll-Like Receptor 9 Ligand)

Changes in lipid metabolism have been implicated in protection against infectious diseases. In the first experiment of this study, we measured clinical lipid parameters in a murine model where the unmethylated cytidine phosphate guanosine (CpG) oligodinucleotide (ODN1826), a Toll-like receptor 9 (TLR9) agonist was administered in combination with D-galactosamine (GalN) that caused relatively liver-specific inflammation and toxicity. In the control mice group injected with phosphate-buffered saline (PBS) (acute psychological stress model associated with blood sampling), the serum triglyceride (TG) levels showed a rapid decrease followed by a rebound at 24 h as we have recently reported. However, such a TG rebound was impaired in the CpG/GalN- and solely CpG-treated groups of mice despite an absence of liver injury based on serum alanine aminotransferase levels in the latter group. Thus, the stress-associated serum TG rebound was abrogated by the injection of a sub-hepatotoxic CpG dose. In the second experiment, we simply measured the hepatic CD36 and SACRB1 (the gene for scavenger receptor B1 (SR-B1)) transcripts after the i.p. administration of PBS, CpG or CpG/GalN. There was a remarkable elevation of hepatic CD36 transcript expression in both the CpG- and CpG/GalN-treated mice at 8 h post-CpG injection whereas the increase in the PBS-treated mice was slower than the former two groups, suggesting that hepatic CD36 transcript expression is more pronounced in the combined stress models than under psychological stress alone. The individual mice data showed that the increase in CD36 expression was accompanied by a reduction in SCARB1 mRNA, showing reciprocal regulation between these two genes. Together with our previously reported findings, these data suggest that, in a murine model combining psychological stress with TLR-triggered hepatic inflammation, the psychological stress facilitates liver uptake of plasma TG (and its components fatty acids), but the subsequent re-esterification and/or release of TG-rich lipoproteins from the liver is impaired due to the concomitant TLR-signaling. We hypothesize that lipid metabolism during acute stress shifts toward an elevated hepatic uptake of lipids due to concomitant TLR signaling, facilitating the clearance of bacterial lipids by the liver.

Toll-like Receptor9在大鼠胰腺表达及与大鼠急性胰腺炎相关性的研究

目的 (1)建立急性胰腺炎大鼠模型,定性检测Toll-like Receptor 9(TLR 9)在大鼠胰腺的表达、分布情况;(2)定量测定TLR 9在大鼠急性胰腺炎不同时间点的表达变化情况;(3)结合TLR 9在大鼠胰腺的组织分布、表达情况及在雨蛙素诱导性胰腺炎(cerulein-induced pancreatitis,CIP)早期24 h的表达改变,探讨TLR9与CIP发生发展的相关性.方法(1)采用Wistar大鼠,并随机分配进入实验组或对照组;通过皮下注射雨蛙素建立急性胰腺炎模型;(2)采用免疫组化方法检测TLR 9在正常大鼠胰腺及CIP时大鼠胰腺的表达TLR 9在大鼠胰腺的组织分布情况;(3)提取总RNA,采用实时荧光定量逆转录-多聚酶链反应(Quantitative-Real-Time;QRT-PCR)法测定TLR9基因的表达.(4)分析TLR9的分布特征及可能的意义(5)统计分析TLR 9 mRNA的表达情况与CIP发生、发展的关系.结果 (1)TLR 9主要分布于胰管上皮、血管内皮和胰岛;(2)外分泌腺泡细胞没有明显的表达;(3)QRT-PCR结果显示TLR9 mRNA在正常大鼠胰腺组织呈现低水平表达;(4)CIP早期TLR9 mRNA表达出现快速上调并在1 h时达到最高值;TLR 9 mRNA表达在CIP前4 h内维持于高水平;其后下降缓慢,至到CIP的第24小时也未降至正常,保持相对较高的表达水平.结论 (1)TLR 9在大鼠胰腺有表达,且表达具有一定的组织特异性;(2)TLR9在CIP胰腺组织中的表达明显升高,提示TLR 9在胰腺炎早期炎症反应的发生、发展中具有重要作用,与之存在相关性.

Epithelial toll-like receptor 9 signaling in colorectal inflammation and cancer: Clinico-pathogenic aspects

Toll-like receptors (TLRs) recognize specific motifs which are frequently present in bacteria, fungi, prokaryotes and viruses. Amongst TLRs, TLR9 can be activated by such bacterial or viral DNA fragments, immunoglobulin-DNA complexes or synthetic oligonucleotides, which all contain unmethylated cytosineguanine nucleotide sequences (CpGs). Emerging data indicate that TLR9 signaling has a role in, and may influence, colorectal carcinogenesis and colonic inflammation. CpGs are classified into three groups according to their influence on both the antigen-specific humoraland cellular immunity, and the production of type 1 interferons and proinflammatory cytokines. TLR9 activation via CpGs may serve as a new therapeutic target for several cancerous and various inflammatory conditions. Due to its probable anti-cancer effects, the application possibilities of TLR9-signaling modulation may be extremely diverse even in colorectal tumors. In this review we aimed to summarize the current knowledge about TLR-signaling in the pathogenesis and therapy of inflammatory bowel diseases and colorectal cancer. Due to the species-specific differences in TLR9 expression, however, one must be careful in translating the animal model data into the human system, because of the differences between CpG-oligodeoxynucleotide-responsive cells. TLR9 agonist DNA-based immunomodulatory sequences could also represent a promising therapeutic alternative in systemic inflammatory conditions and chronic colonic inflammations as their side effects are not significant.

Toll-like receptor 9 polymorphisms and Helicobacter pylori influence gene expression and risk of gastric carcinogenesis in the Brazilian population

BACKGROUND Toll-like receptors(TLRs)are the first line of host defense,and are involved in Helicobacter pylori(H.pylori)recognition and activation of both inflammatory and carcinogenic processes.The presence of single nucleotide polymorphisms(SNPs)in genes that activate the immune response may modulate the risk of precancerous lesions and gastric cancer(GC).Among them,Toll-like receptor 9(TLR9)polymorphisms have emerged with a risk factor of infectious diseases and cancer,however the studies are still inconclusive.AIM To evaluate whether TLR9 rs5743836 and rs187084 SNPs contribute to the risk of gastric carcinogenesis,and its influence on mRNA expression.METHODS A case-control study was conducted to evaluate two TLR9 SNPs(TLR9-1237 TCrs5743836 and TLR9-1486 CT-rs187084)in chronic gastritis(CG)and GC patients.A total of 609 DNA samples of peripheral blood[248 CG,161 GC,and 200 samples from healthy individuals(C)]were genotyped by polymerase chain reaction-restriction fragment length polymorphism.All samples were tested for the H.pylori infection using Hpx1 and Hpx2 primers.Quantitative polymerase chain reaction by TaqMan?assay was used to quantify TLR9 mRNA from fresh gastric tissues(48 GC,26 CG,and 14 C).RESULTS For TLR9-1237,the TC+CC or CC genotypes were associated with a higher risk of GC than C[recessive model odds ratio(OR)=5.01,95%confidence interval(CI):2.52-9.94,P<0.0001],and the CG(recessive model OR=4.63;95%CI:2.44-8.79,P<0.0001)groups.For TLR9-1486,an association between the CT+TT genotypes and increased risk of both GC(dominant model OR=2.72,95%CI:1.57-4.72,P<0.0001)and CG(dominant model OR=1.79,95%CI:1.15-2.79,P=0.0094)was observed when compared to the C group.Moreover,the presence of TLR9-1237 TC/CC+TLR9-1486 CC genotypes potentiate the risk for this neoplasm(OR=18.57;95%CI:5.06-68.15,P<0.0001).The TLR9 mRNA level was significantly higher in the GC group(RQ=9.24,P<0.0001)in relation to the CG group(RQ=1.55,P=0.0010)and normal mucosa(RQ=1.0).When the samples were grouped according to the polymorphic genotypes and the presence of H.pylori infection,an influence of TLR9-1237 TC+CC polymorphic genotypes(P=0.0083)and H.pylori infection(P<0.0001)was observed on the upregulation of mRNA expression.CONCLUSION Our findings show that TLR9 rs5743836 and rs187084 polymorphisms are associated with a higher risk of carcinogenesis gastric,and that TLR9 mRNA levels can be modulated by TLR9-1237 TC+CC variant genotypes and H.pylori infection.

Expression of Toll-like Receptor 9 in Peripheral Blood Mononuclear Cells from Patients with Different Hepatitis B and C Viral Loads

The aim of the present study was to investigate the expression of toll-like receptors （TLR） 9 in peripheral blood mononuclear cells （PBMC） of patients with chronic hepatitis B and C with different virus copies. The study group included 90 patients （60 with chronic hepatitis B, and 30 with chronic hepatitis C）, and 20 healthy people served as control group. The protein and mRNA levels of TLR9 were detected by using flow cytometry and real-time PCR. The serum viral copies of HBV and HCV were measured in all patients, and the correlation between HBV-DNA copies or HCV-RNA copies and the TLR9 expression was analyzed. Our results demonstrated that HBV or HCV infection led to a decreased expression of TLR9 mRNA and protein compared to the control group （P〈0.05）. The TLR9 protein and mRNA levels were negatively correlated with serum viral copies of HBV and HCV （r=-0.632, r=-0.909, P〈0.01）. It was concluded that TLR9 mRNA and protein are down-regulated in PBMC of HBV-infected or HCV-infected patients, and they are negatively correlated with serum viral copies and play an important role in detecting viral replication of HBV and HCV.

Toll-like receptor 9 gene mutations and polymorphisms in Japanese ulcerative colitis patients

Abnormal innate immune responses toward luminal bacteria play an important role in the pathogenesis of inflammatory bowel disease.It has been demonstrated that bacteria having CpG DNA ameliorate experimental colitis in mice,and Toll-like receptor 9 (TLR9) signaling mediates the anti-inflammatory effects in mouse colonic inflammation.A gene variation in NOD2/CARD15 has been reported in Crohn's disease (CD) patients in Western countries,but this variation has not been identified in Japanese CD patients.Therefore,we hypothesized that TLR9 is a key factor in the development of ulcerative colitis (UC),and we investigated gene mutations and polymorphisms of TLR9 in Japanese UC patients.Three single nucleotide polymorphisms (SNPs) in TLR9 were identified in healthy controls,and were assessed in 48 UC patients and 47 healthy controls.Control subjects were matched for age,sex and date of blood sampling from among a subgroup of participants.We found that TLR9-1486CC,1174GG and 2848AA increase the risk of UC [odds ratio (OR) 2.64,95% confidence interval (95% CI):1.73-6.53,P=0.042],and TLR9-1486TT,1174AA and 2848GG decrease the risk of UC (OR 0.30,95% CI:0.10-0.94,P=0.039),although there were no correlations between SNPs and disease phenotype or TLR9 mRNA expression.These findings suggest that TLR9 polymorphisms are associated with increased susceptibility to UC.

Activation of Toll-like receptors signaling in non-small cell lung cancer cell line induced by tumor-associated macrophages

Background： Inflammation is often linked with the progress and poor outcome of lung cancer. The understanding of the relationship between tumor-associated macrophages （TAMs） and lung cancer cells involves in the underlying mechanism of inflammatory cytokine production. Toll-like receptors （TLRs） are engaged in promoting the production of pro-inflammatory cytokines and play an important role in tumor immunology. Methods： To investigate the mechanisms by which TAMs influence the production of pro-inflammatory cytoldnes in lung cancer cells, we established an in vitro coculture system using TAMs and human non- small cell lung cancer （NSCLC） cell line SPC-A1. Levels of interleukin （IL）-113, IL-6 and IL-8 in SPC-A1 were evaluated by RT-PCR and cytometric bead array assay after being cocultured with TAMs. Expression changes of TLRs and TLRs signaling pathway proteins in SPC-Al were further confirmed by RT-PCR and western blot. The level changes of IL-1β, IL-6 and IL-8 in SPC-Al were also detected after the stimulation of TLRs agonists. Results： We found that the phenotype markers of TAMs were highly expressed after stimulating human monocyte cell line THP-1 by phorbol-12-myristate-β-acetate （PMA）. Higher mRNA and supernate secretion levels of IL-1β, IL-6 and IL-8 were detected in SPC-A1 after being eocultured with TAMs. We also found that TLR1, TLR6 and TLR7 were up-regulated in SPC-A1 in the coculture system with TAMs. Meanwhile, TLRs signaling pathway proteins were also significantly activated. Moreover, pre-treatment with agonist ligands for TLR1, TLR6 and TLR7 could dramatically promote inductions of IL-1β, IL-6 and IL-8. Conclusions： These findings demonstrated that TAMs may enhance IL-1β, IL-6 and IL-8 expressions via TLRs signaling pathway. We conclude that TAMs contribute to maintain the inflammation microenvironment and ultimately promote the development and progression of lung cancer.

Science Letters:A synthetic Toll-like receptor 2 ligand decreases allergic immune responses in a mouse rhinitis model sensitized to mite allergen

It has been proposed that activation of Toll-like receptors （TLRs） plays crucial roles in the polarization of adaptive immune responses. A synthetic Toll-like receptor 2 （TLR2） ligand, Pam3CSK4, has been reported to modulate the balance of Th1/Th2 responses. We evaluated the modulation effect of Pam3CSK4 on allergic immune response in a mouse rhinitis model sensitized to house dust mite allergen （HDM）. Mice were sensitized and challenged with Dermatophagoidesfarinae allergen （Der f）, and then the allergic mice were treated by Pam3CSK4. Nasal allergic symptoms and eosinophils were scored. Der f-specific cytokine responses were examined in the splenocytes and bronchoalveolar lavage fluid （BALF）. Serum level of total IgE was also detected. After establishing a mouse allergic rhinitis model with HDM, we have showed that Pam3CSK4 treatment not only ameliorated the nasal allergic symptoms remarkably but also decreased the eosinophils and total inflammation cells in BALF significantly. Analysis of cytokine profile found that IFN-7 released from either BALF or stimulated splenocytes increased markedly in Pam3CSK4-treated mice, while IL-13 decreased significantly. Moreover, serum level of total IgE was significantly lower in Pam3CSK4-treated mice than in the untreated. Thus, in an allergic rhinitis mouse model developed with HDM, Pam3CSK4 was shown to exhibit an antiallergic effect, indicating its potential application in allergic diseases.

Effect of remifentanil on toll-like receptor 4, NF-κB and IL-6 in rabbit myocardial ischemia/reperfusion model

Objective： To investigate whether remifentanil induced cardioprotecting effect is associated with expression of toll-like receptor 4 （TLR4）, nuclear factor rB （NF-r.B） and serum interleukin -6 （IL-6）. Methods： Fifty rabbits were randomly divided into 5 groups （n=10） according to the treatment： sham operation group （group A）, ischemla-reperfusion group （group B）, low-dose remifentanil group （group C）, mediate-dose remifentanil group （group D）, and high-dose remlfentanil group （group E） Myocardial TLR4 mRNA levels, NF-r.B protein expression and serum levels of IL-6 were observed in 120 min after reperfusion. Results： The myocardial expressions of TLR4 mRNA, NF-rd3 protein and IL-6 level in sera of groups B, C, D and E were elevated compared with group A. However, remifentanil significantly reduced the levels of TLR4 mRNA, NF- r.B protein expression and serum IL-6 in groups C, D and E compared with group B. There were remarkable differences between the groups （P〈O.O1）. Conclusion： Intravenous remifentanil has protective effect against rabbit myocardial ischemia/reperfusion injury. This effect may be associated with TLR4, NF-r.B expressions on myocytes and serum level of IL-6 in a dose-dependent manner

Effects of Klebsiella pneumoniae on Toll-Like Receptor-Dependent Endoplasmic Reticulum Stress-Related Signaling Pathways and Gene Expression and Promotes HLA-B27 Misfolding

Klebsiella has been considered as initiator of AS （ankylosing spondylitis） for nearly four decades. This study aimed to demonstrate that Klebsiella triggers ERS （endoplasmic reticulum stress） and HLA-B27 heavy chain misfolding. CA46 cells or splenocytes obtained from wild-type, MyD88/ or TLR9/ mice were stimulated with KP （Klebsiella pneumoniae） or its components including CPS （capsule polysaccharide）, LPS （lipopolysaccharide）, and KP gDNA （genomic deoxyribonucleic acid） respectively for 24 h and 48 h. The activation of ERS-related signaling was detected by Western blotting or RT-PCR, and the level of misfolded HLA-B27 was determined by non-reducing protein gel electrophoresis and Western blotting. The protein expression of BiP/Grp78 and calreticulin, the alternative splicing of XBP-1 mRNA （messenger ribonucleic acid）, and the activation of caspase-12 and p38 were increased in a dose-dependent manner in HLA-B27-expressing CA46 cells after treatment with decapsulated KP. We also demonstrate that the EP, S-inducing effects occur via the TLR （Toll-like receptor）/MyD88-dependent signaling pathway. Significantly, HLA-B27 misfolding was also detected in decapsulated KP-treated B27-expressing cells. These results suggest that the non-antigen-specific induction of ERS and B27 misfoiding through TLR/MyD88 signaling might promote KP antigen-initiated autoreactive responses via the presentation of misfolded B27, and that small-molecules targeting TLRs might have potential as novel therapeutic agents for AS.

TLR9基因rs 187084位点多态性与子宫内膜异位症的相关性研究

目的：探讨Toll样受体（Tol- like receptors/TLRs）9基因rs187084位点多态性与子宫内膜异位症（EMs）发病风险的关系。方法采用DNA直接测序法检测104例EMs患者（EMs组）和100例非EMs患者（对照组）的TLR9基因rs187084位点的多态性，比较各基因型和等位基因的分布情况。结果 EMs组和对照组中，TLR9基因rs187084位点的AA、AG、GG3种基因型频率分别为42.30%、28.85%、28.85%和52.00%、34.00%、14.00%，两组比较差异有统计学意义（P=0.036）；A、G等位基因频率分别为56.73%、43.27%和69.00%、31.00%，两组比较差异有统计学意义（P=0.01）；携带GG基因型明显增加EMs的发病风险（OR=1.911，95％CI 1.113～3.282）。结论 TLR9基因rs187084位点多态性与EMs的发病风险相关。

TLR受体9在急性肺损伤大鼠血清、肺灌注液及肺组织中的表达

目的:TLR受体9(TLR-9)在胸部创伤所致急性肺损伤(ALI)大鼠血清、肺灌注液及肺组织中表达。方法:40只雄性SD大鼠随机分为正常对照组(8只)和ALI模型组(32只),ALI模型组采用BIM-Ⅱ型水平式生物撞击机持续撞击大鼠的右侧胸部造模,于造模后5、24、48和72 h处死大鼠,取血清及肺组织;计算各组大鼠肺组织的湿/干重比(W/D),HE染色观察各组肺组织变化,ELISA法检测血清及肺泡灌洗液中TLR-9水平,免疫组化染色检测肺组织中TLR-9和IL-6的表达,RT-PCR法检测IL-6 mRNA及TLR9 mRNA的变化。结果:与正常对照组比较,ALI模型组大鼠肺水肿、肺组织充血、中性粒细胞浸润、炎性渗出较明显;与正常对照组比较,ALI模型组造模48、72 h时大鼠肺组织W/D降低,血清及肺泡灌洗液中TLR-9蛋白的表达增高,肺组织中TLR-9、IL-6蛋白和mRNA的表达升高,且造模48 h时达最高,差异有统计学意义(P<0.05)。结论:TLR9可能与胸部创伤所致大鼠ALI时肺部炎症因子的分泌有关。

Effects of CPG ODN on biological behavior of PANC-1 and expression of TLR9 in pancreatic cancer

AIM:To determine the expression of toll-like receptor 9(TLR9) in pancreatic tumor and the effects of cytosine phosphate-guanosine oligodeoxynucleotides 2216(CPG ODN2216) on biological behavior of pancreatic carcinoma cell line PANC-1 and explore their clinical significance.METHODS:The immunohistochemistry and Western blot were used to determine the expression of TLR9 protein in pancreatic cancer tissues,and immunofluorescence staining was performed to detect the TLR9 protein expression in pancreatic carcinoma cell line PANC-1.To assess the effects of CPG ODN2216 on the invasive property of Panc-1 cells,in vitro cell adhesion,wound-healing scrape,and invasion and cell colony formation were evaluated.RESULTS:TLR9 was highly expressed in pancreaticcancer tissues and PANC-1 cells.The percentage of positive cells expressing TLR9 protein in human pancreatic tissues,paracancerous tissues and normal tissues were 73.3%,33.3% and 20.0%,respectively,and the protein expression level of TLR9 was gradually descending(P < 0.05).In vitro tests in wound-healing scrape,cell adhesion,colony formation and matrigel invasion showed that the adhesion and motility of PANC-1 cells in CPG ODN 2216 treatment group were signif icantly lower than in the control group(P < 0.05).The cell growth assay showed that the proliferative ability of PANC-1 cells in treatment group was significantly decreased and CPG ODN2216 had an inhibitive effect in the growth of Panc-1 cells in a dose and time-dependent manner(P < 0.05).CONCLUSION:The gene of TLR9 is correlated with the invasive and metastatic potential of human pancreatic carcinoma,and CPG ODN2216 induces the inhibition of migration and invasion of Panc-1 cells.

TLR9 Expression and Its Role in Chemosensitivity to DDP in Human Cervical Cancer Cells in vitro

Inflammation and infection play an important role in the pathogenesis of many cancers.Toll-like receptors（TLRs） are a class of pattern recognition receptors that recognize conserved components of microbes and trigger the immune response against invading microorganisms.Toll-like receptor 9（TLR9） recognizes non-methylated cytosine-phosphateguanosine（CpG） DNA sequences which are the surrogate for viral DNA.TLR9 may react to tumor development and progression during chronic inflammation that involves the tumor microenvironment.In order to study the role of TLR9 in cervical cancer,we analyzed the TLR9 expression in different types of HPV infection cervical cancer cells.Then we detected if CpG sequences influenced the TLR9 expression and the sensitivity to cisplatin（DDP） of these cervical cancer cells in vitro.The expression of TLR9 mRNA and protein in SiHa,Hela and C33A cells was detected by RT-PCR and Western blotting.Real-time PCR was used to examine the TLR9 expression changes induced by CpG.Chemosensitivity of the cervical cancer cells to cisplatin（DDP） was measured by MTT.It was observed that the expression of TLR9 mRNA and protein was increased gradually in SiHa（HPV16＋）,Hela（HPV18＋） and C33A（HPV-） cells.Low doses of CpG increased the TLR9 expression only in C33A（HPV-） cells,but not in SiHa（HPV16＋） and Hela（HPV18＋） cells.Furthermore,low dose of CpG significantly increased the sensitivity of C33A（HPV-） cells,but not that of SiHa（HPV16＋） and Hela（HPV18＋） cells.These results indicated that TLR9 may serve as a protective agent in HPV negative cervical cancer cells.It was concluded that TLR9 could improve the sensitivity to DDP in HPV negative cervical cancer cells and might represent a potential therapeutic option in clinical practice.

Recombinant E.coli LLO/OVA Induces Murine BMDCs Maturation via TLR4 and NOD1 Receptor and Promotes Specific Cytotoxic T Cell Immunity

Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells （BMDCs） and T lymphocytes in vitro.Methods After BMDCs stimulated by E.coli LLO/OVA,their Toll-like receptor （TLR） and nucleotide-binding oligomerization domain （NOD） receptor signalling pathway were examined by superarray hybridization;and the priming effect of the vaccine activated BMDCs on CD4＋T and CD8＋T was determined by [3H]thymidine uptake and ELISA,the tumor cytotoxic effect of activated CD8＋T cells was determined by cytotoxic assay.Results After BMDCs were activated by E.coli LLO/OVA via TLR4,NOD1 receptor and NF-κB signalling pathway,the expression of their surface molecules including MHC class Ⅰ,MHC class Ⅱ,CD40,CD80 and CD86 significantly up-regulated;the secretion of IL-12 and IFN-？ increased also.The mature BMDCs stimulated the allergic CD4＋T and CD8＋T cells proliferation and their IL-2 and IFN-γ secretion,and the activated CD8＋T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.Conclusion E.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.

CopE and TLR6 RNAi-mediated tomato resistance to western flower thrips

The western flower thrips(WFT;Frankliniella occidentalis)is a mesophyll cell feeder that damages many crops.Management of WFT is complex due to factors such as high fecundity,short reproduction time,ability to feed on a broad range of host plants,and broad pesticide resistance.These challenges have driven research into developing alternative pest control approaches for WFT.This study analyzed the feasibility of a biological control-based strategy to manage WFT using RNA interference(RNAi)-mediated silencing of WFT endogenous genes.For the delivery of RNAi,we developed transgenic tomato lines expressing double-stranded RNA(dsRNA)of coatomer protein subunit epsilon(CopE)and Toll-like receptor 6(TLR6)from WFT.These genes are involved in critical biological processes of WFT,and their dsRNA can be lethal to these insects when ingested orally.Adult WFT that fed on the transgenic dsRNAexpressing tomato flower stalk showed increased mortality compared with insects that fed on wild-type samples.In addition,WFT that fed on TLR6 and CopE transgenic tomato RNAi lines showed reduced levels of endogenous CopE and TLR6 transcripts,suggesting that their mortality was likely due to RNAi-mediated silencing of these genes.Thus,our findings demonstrate that transgenic tomato plants expressing dsRNA of TLR6 and CopE can be lethal to F.occidentalis,suggesting that these genes may be deployed to control insecticide-resistant WFT.

Toll-like receptor 10 (TLR10) exhibits suppressive effects on inflammation of prostate epithelial cells

Prostate inflammation (PI) is closely related to the development and progression of chronic prostatic diseases: benign prostatic hyperplasia and prostate cancer. Toll-like receptor (TLR) 2 has been reported to be associated with inflammatory diseases, such as infections, autoimmune diseases, and cancers. Meanwhile, TLR10, which can form heterodimers with TLR2, has been considered an orphan receptor without an exact function. The present study therefore aims to examine the effects of TLR2 and TLR10 on PI. Prostate samples and clinical data were obtained from the patients diagnosed with benign prostatic hyperplasia. The inflammatory cell model was established by adding lipopolysaccharide to RWPE-1 cells. Prostate tissues/cells were examined by histological, molecular, and biochemical approaches. Both TLR2 and TLR10 were found to be expressed in prostate tissues and RWPE-1 cells. mRNA/protein expression levels of TLR2 and TLR10 were both positively correlated with prostate tissue inflammatory grades. Lipopolysaccharide-stimulated RWPE-1 cells expressed higher levels of TLR2, TLR10, high mobility group box 1 (HMGB1), phosphonuclear factor kappa-light-chain-enhancer of activated B-cells P65 (phospho-NF-κB P65), interleukin (IL)-6, and IL-8 than control cells. Moreover, HMGB1, phospho-NF-κB P65, IL-6, and IL-8 were down regulated after TLR2 knockdown and upregulated after TLR10 knockdown in RWPE-1 cells. TLR2 stimulation can activate the inflammatory signaling cascade in prostate epithelial cells. Conversely, TLR10 exhibited suppressive effects on inflammation. With antagonistic functions, both TLR2 and TLR10 were invoIved in PI. TLR10 could be a novel target in modulating inflammatory signal transduction of prostate epithelial cells.

Molecular hydrogen regulates the expression of miR-9,miR-21 and miR-199 in LPS-activated retinal microglia cells

·AIM: To explore the potential mechanism of molecular hydrogen in the regulation of miRNA expression and signal-modulating activities. ·METHODS: Retinal microglia cells were activated by Lipopolysaccharides (LPS) and then treated with hydrogen -saturated medium or normal medium without hydrogen. qRT -PCR was used to detect the expression difference in miR-9, miR-21 and miR-199 between these two groups. Moreover, the expression of LPS -induced signaling proteins, including Myd88, IKK -β, NF -kB, and PDCD4, were detected by Western blotting. ·RESULTS: The results demonstrated a marked downregulation of miR -9 and miR -21 and up -regulation of miR-199 by hydrogen treatment; the expression of Myd88 and IKK-β was decreased after hydrogen treatment, whereas PDCD4 was increased, and there was no significant change in NF-kB expression. · CONCLUSION: The results in the present study indicate that miR -9, miR -199 and miR -21 play an important role in the anti -inflammatory regulation of LPS -activated microglia cells by molecular hydrogen, which will help to explain the protective mechanism of molecular hydrogen against inflammatory injury.·

Toll-like receptor 3(TLR3)regulation mechanisms and roles in antiviral innate immune responses

Toll-like receptor 3(TLR3)is a member of the TLR family,mediating the transcriptional induction of type I interferons(IFNs),proinflammatory cytokines,and chemokines,thereby collectively establishing an antiviral host response.Studies have shown that unlike other TLR family members,TLR3 is the only RNA sensor that is utterly dependent on the Tollinterleukin-1 receptor(TIR)-domain-containing adaptor-inducing IFN-β(TRIF).However,the details of how the TLR3-TRIF signaling pathway works in an antiviral response and how it is regulated are unclear.In this review,we focus on recent advances in understanding the antiviral mechanism of the TRIF pathway and describe the essential characteristics of TLR3 and its antiviral effects.Advancing our understanding of TLR3 may contribute to disease diagnosis and could foster the development of novel treatments for viral diseases.

TLR7/8 signalling affects X-sperm motility via the GSK3α/β-hexokinase pathway for the efficient production of sexed dairy goat embryos

Background:Goat milk is very similar to human milk in terms of its abundant nutrients and ease of digestion.To derive greater economic benefit,farmers require more female offspring(does);however,the buck-to-doe offspring sex ratio is approximately 50%.At present,artificial insemination after the separation of X/Y sperm using flow cytometry is the primary means of controlling the sex of livestock offspring.However,flow cytometry has not been successfully utilised for the separation of X/Y sperm aimed at sexing control in dairy goats.Results:In this study,a novel,simple goat sperm sexing technology that activates the toll-like receptor 7/8(TLR7/8),thereby inhibiting X-sperm motility,was investigated.Our results showed that the TLR7/8 coding goat Xchromosome was expressed in approximately 50%of round spermatids in the testis and sperm,as measured from cross-sections of the epididymis and ejaculate,respectively.Importantly,TLR7/8 was located at the tail of the Xsperm.Upon TLR7/8 activation,phosphorylated forms of glycogen synthase kinaseα/β(GSK3α/β)and nuclear factor kappa-B(NF-κB)were detected in the X-sperm,causing reduced mitochondrial activity,ATP levels,and sperm motility.High-motility Y-sperm segregated to the upper layer and the low-motility X-sperm,to the lower layer.Following in vitro fertilisation using the TLR7/8-activated sperm from the lower layer,80.52±6.75%of the embryos were XX females.The TLR7/8-activated sperm were subsequently used for in vivo embryo production via the superovulatory response;nine embryos were collected from the uterus of two does that conceived.Eight of these were XX embryos,and one was an XY embryo.Conclusions:Our study reveals a novel TLR7/8 signalling mechanism that affects X-sperm motility via the GSK3α/β-hexokinase pathway;this technique could be used to facilitate the efficient production of sexed dairy goat embryos.