Chicken toll-like receptor 7的研究进展及展望

TLRs(toll-like receptors,TLRs)是一种天然免疫分子,它通过识别病原体相关分子模式在其宿主机体的天然免疫系统起着重要的防御作用。chTLR7(Chicken toll-like receptor 7)和其他Toll家族成员一样属于Ⅰ型跨膜蛋白受体,位于鸡的1号染色体上。本文主要探讨了chTLR7的发现及其配体、分布、转导途径、生物学功能及其与细胞凋亡关系论述,另外对其以后的研究进行了展望进而更进一步了解其功能。

The involvement of hypoxia-inducible factor 1 alpha in Toll-like receptor 7/8-mediated inflammatory response

Toll-like receptors （TLRs） 7 and 8 are crucial in host defence against single-stranded RNA （ssRNA） viruses. Such viruses cause severe illnesses, which remain a serious medical burden in both industrialised and developing countries. TLR7/8 downstream signaling leads tO a dramatic cellular stress associated with energy consumption. However, the molecular mechanisms of cell survival and adaptation to TLR7/8-induced stress, which give the cells an opportunity to initiate proper inflammatory reactions, are not clear at all. Here we report for the first time that ligand-induced activation of TLR7/8 leads to the accumulation of hypoxia-inducible factor 1 alpha （HIF-1α） protein in THP-1 human myeloid macrophages via redoxand reactive nitrogen species-dependent mechanisms. MAP kinases and phosphoinositol-3K are not involved in TLR7/8-mediated HIF-1α accumulation. Experiments with HIF-1α knockdown THP- 1 cells have clearly demonstrated that HIF-1α is important for the protection of these cells against TLR7/8-induced depletion of ATP. Thus, HIF-1α might support both cell survival and the production of pro-inflammatory cytokines upon TLR7/8 activation.

MicroRNA-630 alleviates inflammatory reactions in rats with diabetic kidney disease by targeting toll-like receptor 4

BACKGROUND Diabetic kidney disease(DKD)is a major complication of diabetes mellitus.Renal tubular epithelial cell(TEC)damage,which is strongly associated with the inflammatory response and mesenchymal trans-differentiation,plays a significant role in DKD;However,the precise molecular mechanism is unknown.The recently identified microRNA-630(miR-630)has been hypothesized to be closely associated with cell migration,apoptosis,and autophagy.However,the association between miR-630 and DKD and the underlying mechanism remain unknown.AIM To investigate how miR-630 affects TEC injury and the inflammatory response in DKD rats.METHODS Streptozotocin was administered to six-week-old male rats to create a hypergly cemic diabetic model.In the second week of modeling,the rats were divided into control,DKD,negative control of lentivirus,and miR-630 overexpression groups.After 8 wk,urine and blood samples were collected for the kidney injury assays,and renal tissues were removed for further molecular assays.The target gene for miR-630 was predicted using bioinformatics,and the association between miR-630 and toll-like receptor 4(TLR4)was confirmed using in vitro investigations and double luciferase reporter gene assays.Overexpression of miR-630 in DKD rats led to changes in body weight,renal weight index,basic blood parameters and histopathological changes.RESULTS The expression level of miR-630 was reduced in the kidney tissue of rats with DKD(P<0.05).The miR-630 and TLR4 expressions in rat renal TECs(NRK-52E)were measured using quantitative reverse transcription polymerase chain reaction.The mRNA expression level of miR-630 was significantly lower in the high-glucose(HG)and HG+mimic negative control(NC)groups than in the normal glucose(NG)group(P<0.05).In contrast,the mRNA expression level of TLR4 was significantly higher in these groups(P<0.05).However,miR-630 mRNA expression increased and TLR4 mRNA expression significantly decreased in the HG+miR-630 mimic group than in the HG+mimic NC group(P<0.05).Furthermore,the levels of tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β),and IL-6 were significantly higher in the HG and HG+mimic NC groups than in NG group(P<0.05).However,the levels of these cytokines were significantly lower in the HG+miR-630 mimic group than in the HG+mimic NC group(P<0.05).Notably,changes in protein expression were observed.The HG and HG+mimic NC groups showed a significant decrease in E-cadherin protein expression,whereas TLR4,α-smooth muscle actin(SMA),and collagen IV protein expression increased(P<0.05).Conversely,the HG+miR-630 mimic group exhibited a significant increase in E-cadherin protein expression and a notable decrease in TLR4,α-SMA,and collagen IV protein expression than in the HG+mimic NC group(P<0.05).The miR-630 targets TLR4 gene expression.In vivo experiments demonstrated that DKD rats treated with miR-630 agomir exhibited significantly higher miR-630 mRNA expression than DKD rats injected with agomir NC.Additionally,rats treated with miR-630 agomir showed significant reductions in urinary albumin,blood glucose,TLR4,and proinflammatory markers(TNF-α,IL-1β,and IL-6)expression levels(P<0.05).Moreover,these rats exhibited fewer kidney lesions and reduced infiltration of inflammatory cells.CONCLUSION MiR-630 may inhibit the inflammatory reaction of DKD by targeting TLR4,and has a protective effect on DKD.

METTL5 promotes cell proliferation,invasion,and migration by up-regulating Toll-like receptor 8 expression in colorectal cancer

BACKGROUND N6-methyladenosine(m6A)modification represents the predominant alteration found in eukaryotic messenger RNA and plays a crucial role in the progression of various tumors.However,despite its significance,the comprehensive investigation of METTL5,a key m6A methyltransferase,in colorectal cancer(CRC)remains limited.AIM To investigate the role of METTL5 in CRC.METHODS We assessed METTL5 expression levels in clinical samples obtained from CRC patients as well as in CRC cell lines.To elucidate the downstream targets of METTL5,we performed RNA-sequencing analysis coupled with correlation analysis,leading us to identify Toll-like receptor 8(TLR8)as a potential downstream target.In vitro functional assessments of METTL5 and TLR8 were conducted using CCK-8 assays,scratch assays,as well as assays measuring cell migration and invasion.RESULTS Our findings reveal a pronounced upregulation of METTL5 expression in both CRC cells and tissues,which correlated significantly with an unfavorable prognosis.In vitro experiments unequivocally demonstrated the oncogenic role of METTL5,as evidenced by its promotion of CRC cell proliferation,invasion,and migration.Notably,we identified TLR8 as a downstream target of METTL5,and subsequent down-regulation of TLR8 led to a significant inhibition of CRC cell proliferation,invasion,and tumor growth.CONCLUSION The heightened expression of METTL5 in CRC is strongly associated with clinicopathological features and a poor prognosis,thereby underscoring its potential utility as a critical marker for facilitating early diagnosis and prognostication in CRC.

Toll-like receptors 2 polymorphism is associated with psoriasis: A case-control study in the northern Chinese population

Background:Psoriasis is a disease caused by genetics and immune system dysfunction,affecting the skin and joints.Toll-like receptors(TLRs)play an important role in triggering the innate immune response and controlling adaptive immunity.The role of TLR2 in the progression of psoriasis is not well understood.Methods:A case-control study was conducted on a northern Chinese Han population,consisting of psoriasis patients and healthy control subjects.Genotyping was performed using the tetra-primer amplification refractory mutation system-polymerase chain reaction(ARMS-PCR),and allele and genotype frequencies of four SNPs in TLR2 were analyzed in 270 psoriasis patients and 246 healthy controls.Results:Four TLR2 SNPs(rs11938228,rs4696480,rs3804099,rs5743699)were genotyped and found to be in linkage disequilibrium.The genotype distributions of rs11938228 and rs4696480 in two groups were in Hardy-Weinberg equilibrium and statistically significant except for the overdominance model.The haplotypes ATTC and ATCC were found to be protective against psoriasis.Conclusion:Our study found a correlation between TLR2 genetic variations and the likelihood of psoriasis in northern China.

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.

Three-dimensional common-feature hypotheses for Toll-like receptor 7 agonists

Toll-like receptor 7 （TLR7）, the best known TLRs, has been demonstrated to be useful in fighting against infectious disease. In our study, three-dimensional （3D） pharmacophore models were constructed from a set of 5 TLR7 agonists. Among the 10 common-featured models generated by program Discovery Studio/HipHop, a hypothesis （Hypo2） including one hydrogen-bond donor （D）, one hydrogen-bond acceptor （A）, and two hydrophobic （H） features was considered to be important in evaluating the ligands with TLR7 agonistic activity. The obtained pharmacophore model was further validated using a set of test molecules and the Catalyst TLR7-agonist-subset database. Hypo2 has been shown to identify a range of highly potent TLR7 agonists. Finally, the obtained pharmacophore was further validated using docking studies. Taken together, this model can be utilized as a guide for future studies to design the structurally novel TLR7 agonists.

Toll-like receptor 7 deficiency suppresses type 1 diabetes development by modulating B-cell differentiation and function

Innate immunity mediated by Toll-like receptors(TLRs),which can recognize pathogen molecular patterns,plays a critical role in type 1 diabetes development.TLR7 is a pattern recognition receptor that senses single-stranded RNAs from viruses and host tissue cells;however,its role in type 1 diabetes development remains unclear.In our study,we discovered that Tlr7-deficient(Tlr7^(−/−))nonobese diabetic(NOD)mice,a model of human type 1 diabetes,exhibited a significantly delayed onset and reduced incidence of type 1 diabetes compared with Tlr7-sufficient(Tlr7^(+/+))NOD mice.Mechanistic investigations showed that Tlr7 deficiency significantly altered B-cell differentiation and immunoglobulin production.Moreover,Tlr7^(−/−)NOD B cells were found to suppress diabetogenic CD4^(+)T-cell responses and protect immunodeficient NOD mice from developing diabetes induced by diabetogenic T cells.In addition,we found that Tlr7 deficiency suppressed the antigen-presenting functions of B cells and inhibited cytotoxic CD8^(+)T-cell activation by downregulating the expression of both nonclassical and classical MHC class I(MHC-I)molecules on B cells.Our data suggest that TLR7 contributes to type 1 diabetes development by regulating B-cell functions and subsequent interactions with T cells.Therefore,therapeutically targeting TLR7 may prove beneficial for disease protection.

Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect

The noble gas argon has the potential to protect neuronal cells from cell death.So far,this effect has been studied in treatment after acute damage.Preconditioning using argon has not yet been investigated.In this study,human neuroblastoma SH-SY5Y cells were treated with different concentrations of argon(25%,50%,and 74%;21%O_(2),5%CO_(2),balance nitrogen)at different time intervals before inflicting damage with rotenone(20μM,4 hours).Apoptosis was determined by flow cytometry after annexin V and propidium iodide staining.Surface expressions of Toll-like receptors 2 and 4 were also examined.Cells were also processed for analysis by western blot and qPCR to determine the expression of apoptotic and inflammatory proteins,such as extracellular-signal regulated kinase(ERK1/2),nuclear transcription factor-κB(NF-κB),protein kinase B(Akt),caspase-3,Bax,Bcl-2,interleukin-8,and heat shock proteins.Immunohistochemical staining was performed for TLR2 and 4 and interleukin-8.Cells were also pretreated with OxPAPC,an antagonist of TLR2 and 4 to elucidate the molecular mechanism.Results showed that argon preconditioning before rotenone application caused a dose-dependent but not a time-dependent reduction in the number of apoptotic cells.Preconditioning with 74%argon for 2 hours was used for further experiments showing the most promising results.Argon decreased the surface expression of TLR2 and 4,whereas OxPAPC treatment partially abolished the protective effect of argon.Argon increased phosphorylation of ERK1/2 but decreased NF-κB and Akt.Preconditioning inhibited mitochondrial apoptosis and the heat shock response.Argon also suppressed the expression of the pro-inflammatory cytokine interleukin-8.Immunohistochemistry confirmed the alteration of TLRs and interleukin-8.OxPAPC reversed the argon effect on ERK1/2,Bax,Bcl-2,caspase-3,and interleukin-8 expression,but not on NF-κB and the heat shock proteins.Taken together,argon preconditioning protects against apoptosis of neuronal cells and mediates its action via Toll-like receptors.Argon may represent a promising therapeutic alternative in various clinical settings,such as the treatment of stroke.

Correlation between Toll-like Receptor Gene Polymorphisms and Idiopathic Nephrotic Syndrome in Chinese Children

Objective Idiopathic nephrotic syndrome(INS)is the most common glomerular disease in children.Toll-like receptors(TLRs)have been reported to be associated with response to steroid treatment in children with INS.Nevertheless,the correlation between TLR genes and the progression of INS has not yet been clarified.The present study aimed to investigate the association of single-nucleotide polymorphisms(SNPs)in TLR2,TLR4,and TLR9 with susceptibility to INS as well as the clinical phenotyping of steroid responsiveness in Chinese children with INS.Methods A total of 183 pediatric inpatients with INS were included and given standard steroid therapy.Based on their clinical response to steroids,the patients were classified into three groups:steroid-sensitive nephrotic syndrome(SSNS),steroid-dependent nephrotic syndrome(SDNS),and steroid-resistant nephrotic syndrome(SRNS).A total of 100 healthy children were employed as controls.The blood genome DNA was extracted from each participant.Six SNPs(rs11536889,rs1927914,rs7869402,rs11536891,rs352140,and rs3804099)in TLR2,TLR4,and TLR9 were selected and detected by multiplex polymerase chain reaction with next-generation sequencing to assess TLR gene polymorphisms.Results Among the 183 patients with INS,89(48.6%)had SSNS,73(39.9%)had SDNS,and 21(11.5%)had SRNS.No significant difference was found in the genotype distribution between healthy children and patients with INS.However,the genotype and allele frequencies of TLR4 rs7869402 were significantly different between SRNS and SSNS.Compared with patients with the C allele and CC genotype,patients with the T allele and CT genotype had an increased risk of SRNS.Conclusion TLR4 rs7869402 affected the steroid response in Chinese children with INS.It might be a predictor for the early detection of SRNS in this population.

Fecal microbiota transplantation alleviates experimental colitis through the Toll-like receptor 4 signaling pathway

BACKGROUND Fecal microbiota transplantation(FMT)has shown promising therapeutic effects on mice with experimental colitis and patients with ulcerative colitis(UC).FMT modulates the Toll-like receptor 4(TLR4)signaling pathway to treat some other diseases.However,it remains unknown whether this modulation is also involved in the treatment of UC.AIM To clarify the necessity of TLR4 signaling pathway in FMT on dextran sodium sulphate(DSS)-induced mice and explain the mechanism of FMT on UC,through association analysis of gut microbiota with colon transcriptome in mice.METHODS A mouse colitis model was constructed with wild-type(WT)and TLR4-knockout(KO)mice.Fecal microbiota was transplanted by gavage.Colon inflammation severity was measured by disease activity index(DAI)scoring and hematoxylin and eosin staining.Gut microbiota structure was analyzed through 16S ribosomal RNA sequencing.Gene expression in the mouse colon was obtained by transcriptome sequencing.RESULTS The KO(DSS+Water)and KO(DSS+FMT)groups displayed indistinguishable body weight loss,colon length,DAI score,and histology score,which showed that FMT could not inhibit the disease in KO mice.In mice treated with FMT,the relative abundance of Akkermansia decreased,and Lactobacillus became dominant.In particular,compared with those in WT mice,the scores of DAI and colon histology were clearly decreased in the KO-DSS group.Microbiota structure showed a significant difference between KO and WT mice.Akkermansia were the dominant genus in healthy KO mice.The ineffectiveness of FMT in KO mice was related to the decreased abundance of Akkermansia.Gene Ontology enrichment analysis showed that differentially expressed genes between each group were mainly involved in cytoplasmic translation and cellular response to DNA damage stimulus.The top nine genes correlating with Akkermansia included Aqp4,Clca4a,Dpm3,Fau,Mcrip1,Meis3,Nupr1 L,Pank3,and Rps13(|R|>0.9,P<0.01).CONCLUSION FMT may ameliorate DSS-induced colitis by regulating the TLR4 signaling pathway.TLR4 modulates the composition of gut microbiota and the expression of related genes to ameliorate colitis and maintain the stability of the intestinal environment.Akkermansia bear great therapeutic potential for colitis.

Relationship of Toll-Like Receptors 2 and 4 Gene Polymorphisms with Essential Hypertension in Chinese Han Population

Objective: There are numerous studies suggesting that genetic polymor-phisms of inflammation factors Toll-like receptors 2 and 4 (TLR2, TLR4) might play a role in the pathophysiological process of hypertension. In this study, we evaluated the association in a sample of members of the Chinese Han population. Method: We selected four single nucleotide polymor-phisms (SNP) of TLR2 (rs3804099, rs3804100, rs7656411) and TLR4 (rs1927906) genes, and measured the distributions of genotypic and allelic frequencies in 1063 participants, including 391 essential hypertension pa-tients and 672 controls. Result: No significant differences in the genotypic and allelic frequencies of the four SNPs were detected between cases and controls. However, three haplotypes, CCG, TTG and TTT of TLR2, were significantly associated with a decrease in the risk of essential hyperten-sion (OR: 0.512, 95% CI: 0.397 - 0.660, P P = 0.0038;OR: 0.797, 95% CI: 0.667 - 0.952, P = 0.0122, respectively). Inversely, the risk of essential hypertension increased sig-nificantly in patients with the CTG, TCG or TCT haplotypes (OR: 2.924, 95% CI: 2.157 - 3.963, P P P Conclusion: Our study suggested that haplotypes (CCG, TTG, TTT, CTG, TCG and TCT) of TLR2 might have profound effects on the development of essential hypertension in the Chinese Han population.

Conjugation of toll-like receptor-7 agonist to gastric cancer antigen MG7-Ag exerts antitumor effects

AIM: To investigate the effects of our tumor vaccines on reversing immune tolerance and generating therapeutic response.METHODS: Vaccines were synthesized by solid phase using an Fmoc strategy,where a small molecule toll-like receptor-7 agonist(T7) was conjugated to a monoclonal gastric cancer 7 antigen mono-epitope(T7-MG1) or tri-epitope(T7-MG3).Cytokines were measured in both mouse bone marrow dendritic cells and mouse spleen lymphocytes after exposed to the vaccines.BALB/c mice were intraperitoneally immunized with the vaccines every 2 wk for a total of three times,andthen subcutaneously challenged with Ehrlich ascites carcinoma(EAC) cells.Three weeks later,the mice were killed,and the tumors were surgically removed and weighed.Serum samples were collected from the mice,and antibody titers were determined by ELISA using an alkaline phosphate-conjugated detection antibody for total Ig G.Antibody-dependent cell-mediated cytotoxicity was detected by the lactate dehydrogenase method using natural killer cells as effectors and antibody-labeled EAC cells as targets.Cytotoxic T lymphocyte activities were also detected by the lactate dehydrogenase method using lymphocytes as effectors and EAC cells as targets.RESULTS: Vaccines were successfully synthesized and validated by analytical high performance liquid chromatography and electrospray mass spectrometry,including T7,T7-MG1,and T7-MG3.Rapid inductions of tumor necrosis factor-α and interleukin-12 in bone marrow dendritic cells and interferon γ and interleukin-12 in lymphocytes occurred in vitro after T7,T7-MG1,and T7-MG3 treatment.Immunization with T7-MG3 reduced the EAC tumor burden in BALB/c mice to 62.64% ± 5.55% compared with PBS control(P < 0.01).Six or nine weeks after the first immunization,the monoclonal gastric cancer 7 antigen antibody increased significantly in the T7-MG3 group compared with the PBS control(P < 0.01).As for antibody-dependent cell-mediated cytotoxicity,antisera obtained by immunization with T7-MG3 were able to markedly enhance cell lysis compared to PBS control(31.58% ± 2.94% vs 18.02% ± 2.26%; P < 0.01).As for cytotoxic T lymphocytes,T7-MG3 exhibited obviously greater cytotoxicity compared with PBS control(40.92% ± 4.38% vs 16.29% ± 1.90%; P < 0.01).CONCLUSION: A successful method is confirmed for the design of gastric cancer vaccines by chemical conjugation of T7 and multi-repeat-epitope of monoclonal gastric cancer 7 antigen.

Study on the Relationship between Heat Shock Protein 70 and Toll-like Receptor-4 of Monocytes

Summary: To explore the relation between human heat shock protein 70 (hsp70) and TLR4 in human monocytes in vitro, human monocytes were stimulated with various concentrations of HSP70, and TNF-α production in supernatants was measured by ELISA. Pre-incubated with or without anti-TLR4 mAb, and stimulated with hsp70 (5.0 μg/ml), NF-κB p65 of human monocytes in different time points were detected by immunohistochemistry and monocyte surface expression of TLR4 was measured by flow cytometry. After the human monocytes were pre-incubated with various concentrations of anti-TLR4 and stimulated with hsp70 (5.0 μg/ml), TNF-α production in supernatants was measured. The results showed that hsp70 enhanced NF-κB activation, which was clearly inhibited by anti-TLR4, with the positive cell ratios being 67.44 %, 39.17 %, 31.56 % and 28.05 %, respectively. TLR4 was rapidly down-regulated in the presence of hsp70. MFI of TLR4 on monocytes in different time points were 87.77±5.38, 78.16±6.01 and 45.17±4.97 (P<0.05), 26.98±5.83 (P<0.01), respectively. Moreover, hsp70-induced TNF-α production by human monocytes was inhibited by anti-TLR4. It is suggested that TLR4 is involved in the hsp70-mediated activation of innate immunity.

Effect of Ligands to Toll-Like Receptors (TLR) 3, 7 and 9 on Mice Infected with Mouse Hepatitis Virus A59

Mice infected with mouse hepatitis virus A59 (MHV-A59), an enveloped, positive-strand RNA Co-ronavirus, induce hepatitis, thymus involution, IgG2a-restricted hypergammaglobulinaemia, transaminase release and autoantibodies (autoAb) to liver and kidney fumarylacetoacetate hy-drolase (FAH). Since Toll-like receptors (TLR) play a central role in innate immunity, we explored the effects of TLR3, 7 and 9 stimulation on MHV mouse infection. Thus, the animals were treated with Poly (I:C), Loxoribine and CpG, the respective TLR ligands. MHV-infected mice inoculated with Poly (I:C) had significant lower levels of plasma transaminases and Ig, anti-MHV Ab, and uric acid than MHV-infected animals, whereas autoAb to kidney tissue were observed. Loxoribine only produced a slight decrease of uric acid levels and serum Ig. CpG showed deleterious effects on MHV-infected mice, since survival of animals dramatically dropped to about 10%. AutoAb to murine tissues and uric acid release were not affected, whereas transaminases and anti-MHV Ab were slightly elevated. Besides, CpG administration produced a decrease of the high levels of serum Ig induced by the virus. Therefore, results indicated that TLR3 stimulation appeared to protect the animals against the viral infection, whereas CpG aggravated its signs. Loxoribine, the TLR7 ligand, did not show major effects.

Innate Cytokine Responses and Toll-Like Receptor Induced by Recombinant Porcine Rotavirus VP6 and VP7 Proteins Expressing in <i>Lactobacillus plantarum</i>NC8 Strain Colonization in Mice

The significant function of Toll-like receptors (TLR) is the detection of microbes by host guard cells that guide to the innate immune responses and to the successive adaptive. The current study patterns of TLR2, TLR3 and TLR9 expressing antigen presenting cells (APCs) in blood of mice after colonization with L. plantarum NC8 strain were assessed. The power of L. plantarum on serum innate cytokine and TLR responses stimulated by recombinant NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep and NC8-pSIP409-pgsA were also assessed. We confirmed that L. plantarum NC8 stimulated powerful TLR2 expressing APC responses in blood Recombinant strain stimulated a TLR3 response in spleen, and TLR9 responses were stimulated in blood or in spleen. Recombinant NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep on TLR2 and TLR9 expressing APC responses has a preservative outcome, reliable with the DCpep adjuvant outcome. In serum the recombinant NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep has increased the IL-4 and IFN-γ responses, except that on the TLR3 and TLR9 expressing CD14 APC responses it had an oppressive consequence in spleen and the IFN-α response in serum-stimulated by PRV. Our results give details that following PRV infection after immunization with NC8-pSIP409-pgsA-VP6-DCpep, NC8-pSIP409-pgsA-VP7-DCpep, the systemic TLR2, TLR3, and TLR9 expressing cDC and macrophage/monocyte responses.

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.

人Toll-like receptor 2胞外段的克隆和表达

目的 克隆、表达人Toll-like receptor2(TLR2)胞外段(A26-T588)基因,获得人TLR2胞外段蛋白。方法 RT-PCR扩增TLR2胞外段基因,以pcDNA3.1+质粒为载体在HEK293细胞中表达TLR2胞外段蛋白,同时以pcDNA3.1+/TLR2(A26-T588)重组质粒免疫昆明鼠,制备抗TLR2胞外段蛋白多抗。结果 PCR扩增及重组质粒测序结果表明成功地构建了pcDNA3.1+/TLR2(A26-T588)真核表达质粒,SDS-PAGE分析纯化产物在M_r为68000处出现明显蛋白条带。重组质粒DNA免疫小鼠3次后,血清抗体滴度可达1∶250。TLR2胞外段蛋白可与LPS结合,并在一定的质量浓度范围内呈剂量依赖性。结论 构建的pcDNA3.1+/TLR2(A26-T588)真核表达质粒可在哺乳动物细胞中表达TLR2胞外段蛋白,并与重组质粒DNA免疫小鼠抗血清及TLR2单克隆抗体TL2.1特异性反应,由此证明其表达正确。

广谱模式识别分子Toll-like receptor 2的研究进展

TLR-2（Toll-like receptor 2,TLR-2）是哺乳动物TLRs（Toll-like receptors,TLRs）家族的一员,作为细胞表面的天然受体蛋白,主要参与病原微生物产物的识别及炎症信号传导,介导天然抗感染兔疫;最近又发现其参与机体对非感染因子所致炎性组织损伤的识别。通过对TLR-2参与的识别和细胞内信号传导机制的研究,可为深入探讨抵御微生物感染的机制、对自身正常与非正常组织的识别提供新的思路。

射干制剂对失眠大鼠自主活动及TLR7/MyD88信号通路的影响

目的 探研射干制剂对PCPA致失眠大鼠自主活动、脑电波以及脑组织TLR7、MyD88基因表达的影响。方法 随机将70只健康SD大鼠分为空白对照组、模型对照组、射干制剂低剂量组(26 mg·kg^(-1))、射干制剂中剂量组(52 mg·kg^(-1))、射干制剂高剂量组(104 mg·kg^(-1))、朱砂安神丸组(1.62 g·kg^(-1))、地西泮片组(0.9 mg·kg^(-1))。荧光定量PCR检测各组TLR7、My D88基因的相对表达量;采用Etho Vision大小鼠行为学系统记录大鼠自主活动情况;通过无线遥测系统记录各组睡眠时脑电图。结果 与空白组对照比较,模型对照组脑组织TLR7、MyD88相对表达量显著升高(P<0.01);同模型对照组相比,射干制剂低剂量组、射干制剂中剂量组TLR7、MyD88的mRNA表达量均降低(P<0.05),射干制剂高剂量组TLR7、MyD88 mRNA表达量显著降低(P<0.01);与空白对照组相比,PCPA造模后大鼠运动量明显增加,不同剂量射干制剂组、朱砂安神丸组及地西泮片组运动有不同程度的减少,射干制剂高剂量组运动减少较为明显;给药120 min后,与空白对照组相比,模型对照组脑电波中的δ波百分比降低(P<0.05);与模型对照组相比,射干低中剂量组、朱砂安神丸组、地西泮片组δ波百分比均升高(P<0.05)。结论 射干制剂可有效抑制大鼠兴奋性,增加脑电图δ波百分比,改善睡眠,这些作用可能是通过调节脑组织TLR7和MyD88基因的表达来实现。