细菌脂多糖诱导小鼠DC2.4细胞TLR7蛋白的表达

目的:探讨细菌脂多糖(LPS)对小鼠树突状细胞(DC)系DC2.4 TLR7蛋白表达的诱导作用.方法:用RPMIl640完全培养液培养DC2.4细胞,光学显微镜观察LPS刺激前后的细胞形态特征,RT-PCR和Western blot分别测定TLR7 mRNA和蛋白表达.结果:LPS刺激前后,细胞中均有TLR7 mRNA转录.LPS刺激前,细胞较小而透亮,树突较少,无,TLR7蛋白表达;LPS刺激后,细胞体积增大,树突增粗增多,刺激后12 h、24 h、48 h,TLR7蛋白均有表达,且在3个时间点的表达量基本一致.结论:LPS可诱导DC2.4中TLR7蛋白的表达.     

肝素通过TLR4减少脂多糖刺激的人内皮细胞IL-8表达

目的:观察肝素对脂多糖(lipopolysaccharide, LPS)刺激的人内皮细胞白细胞介素8(interleukin-8,IL-8)水平的影响,并探讨Toll样受体4(Toll-like receptor 4,TLR 4)在其中的可能影响。方法:用LPS(10 mg/L)刺激人肺微血管内皮细胞诱导损伤,肝素治疗组提前15 min分别加入100 U/L及10³ U/L普通肝素,正常对照组加入等量磷酸盐缓冲液。分别在刺激2、6、12 h收集细胞上清,采用酶联免疫吸附法测定上清中IL-8的浓度。在刺激2、6、12 h收集细胞提取RNA,应用实时荧光定量聚合酶链反应检测各组细胞中IL-8、CD14及TLR4 mRNA水平变化。结果:与正常对照组比较,LPS刺激组IL-8 mRNA水平增高,6 h达到高峰,其蛋白水平于12 h达到高峰。LPS刺激下TLR4 mRNA水平增高,6 h达到高峰,肝素降低其水平,差异有统计学意义(P＜0.05)。未检测到CD14 mRNA的表达。结论:LPS刺激下人肺微血管内皮细胞IL-8表达增加。肝素可能通过调节TLR4降低IL-8的水平,从而发挥保护作用。     

体外LPS刺激及CD40的配基化对sCD40基因修饰DCs TLR4-MD2表达及IL-12分泌的影响

目的： 研究体外LPS刺激及CD40的配基化对可溶性CD40（sCD40）基因修饰树突状细胞TLR4-MD2表达及IL-12分泌的影响,为有效利用树突状细胞诱导特异性移植免疫耐受提供实验依据。方法: 脂质体法将质粒pEGFP-N1/sCD40及空质粒pEGFP-N1转染DC2.4细胞株;应用LPS及抗CD40单抗刺激6 h,流式细胞仪检测DC表面TLR4-MD2的表达,RT-PCR法检测DC 的TLR4 mRNA 表达水平,并用ELISA法检测细胞因子IL-12p70的分泌。结果: LPS刺激下调DC表面TLR4-MD2的表达,同时给予CD40配基化可引起TLR4-MD2的表达显著增高;CD40配基化对DC TLR4mRNA 水平表达无影响，但可部分地增高LPS引起的TLR4mRNA 表达降低;此外,CD40的配基化可显著诱导LPS刺激后IL-12分泌增加。sCD40基因修饰DC可拮抗以上作用。结论: 体外LPS及抗CD40单抗刺激下,sCD40基因修饰树突状细胞可显著下调其表面TLR4-MD2的表达,IL-12p70分泌减少，可能与阻断胞浆内的TLR4-MD2的转运过程有关。     

脂多糖刺激对牛单核细胞由来的巨噬细胞Toll样受体表达的影响

目的:研究牛末梢血中单核细胞由来的巨噬细胞在受到LPS刺激后细胞表面Toll样受体表达的变化.方法:试验采取3头日本黑牛外周血,进行分离得到外周血单核细胞,并用Repcell进行巨噬细胞的分离培养,7天后用脂多糖(lipopolysaccharide,LPS)刺激细胞24小时后,通过RT-PCR测定巨噬细胞表面Toll样受体mRNA的表达以及细胞因子mRNA的表达.结果:在LPS刺激24小时后,IL-6、 TNF-α和IL-1βmRNA的表达量显著升高(P＜0.05),与此同时IL-8和IL-12p40 mRNA的表达量相对提高;TLR1和TLR10的转录物显著下降(P＜0.05),然而TLR2、4和6保持稳定.结论:巨噬细胞在受到病原刺激后通过提高IL-6、 TNF-α和IL-1β mRNA的表达量,从而提高了天然免疫的作用.该研究为探讨巨噬细胞在无然免疫中的作用奠定了基础.     

Lewis肺癌细胞TLR4对Foxp3表达的调控

目的:探讨小鼠Lewis肺癌(Lewis lung cancer,LLC)细胞中TLR4对Foxp3表达的调控作用。方法:选择LPS为配体活化TLR4,采用RT-PCR方法检测LPS在不同浓度(0,1,10μg/ml)和不同时间点(12,24,36,48小时)Foxp3 mRNA的表达量的变化,流式细胞术检测有效浓度LPS 10μg/ml和最佳作用时间点24小时Foxp3蛋白和TLR4蛋白表达量的变化,RT-PCR和流式细胞术检测anti-TLR4/MD2抗体阻断TLR4后再用LPS刺激LLC细胞Foxp3表达量的变化。结果:LPS在10μg/ml浓度作用LLC细胞后可显著上调Foxp3 mRNA的表达量,与未刺激组相比差异显著(P<0.05);LPS最佳作用时间为24小时;LPS在10μg/ml浓度作用LLC细胞24小时后可显著上调Foxp3蛋白和TLR4蛋白的表达量,与对照组相比差异显著(P<0.05);阻断TLR4后再用LPS刺激LLC细胞,Foxp3的表达量较未阻断组明显降低(P<0.05)。结论:LLC细胞TLR4蛋白参与对Foxp3的表达调控,TLR4可能是Foxp3的上游信号分子。     

TLR4在LPS诱导的结肠炎症恢复中的作用

目的:研究Toll样受体4(Toll-like receptor 4,TLR-4)在结肠炎症中的作用,探讨LPS在炎症性肠病中的治疗作用。方法:取正常肠上皮细胞进行体外脂多糖(lipopolysaccharide,LPS)干预培养。采用慢病毒转染技术,构建TLR4低表达、正常表达及高表达的肠上皮细胞亚组。正常表达组(normal组)及高表达组(high组)培养基中加入LPS诱导细胞炎症,刺激时间分别为0、2、4 h。Western blot法检测TLR4的表达;收集细胞上清液,ELISA检测各亚组细胞炎症因子TNF-α、IL-6和IL-8的水平。收集细胞,qPCR检测细胞因子TNF-α、IL-6、IL-8、IL-10和IL-1βmRNA的表达水平。划痕试验观察对比2组细胞的迁移能力。结果:LPS干预培养细胞后,TLR4的表达量显著增加(P0.05)。ELISA和qPCR检测高表达组与正常表达组组间细胞因子TNF-α、IL-6、IL-8、IL-10和IL-1β的蛋白和mRNA水平的差异均有统计学意义(P0.05)。划痕试验提示TLR4高表达组的细胞迁移能力明显高于正常对照组。结论:LPS影响TLR4炎症通路的活化,促进前炎症因子及辅助刺激分子的释放,起到调节炎症反应的作用。     

人滋养细胞TLR3信号通路活化抑制血管生成因子表达

目的 研究人类妊娠早期滋养细胞Toll样受体3(TLR3)活化对胎盘血管生成相关因子表达的影响,探讨该通路在妊娠期高血压疾病中的作用.方法 以TLR3配体刺激原代滋养细胞和永生化滋养细胞系swan71,不同时间点收集上清液及细胞.ELISA测定培养上清液中sFlt-1和PIGF浓度,real-time PCR法测定上述分子mRNA表达水平.结果 Poly(I∶C)刺激swan71后24、48和120 h,sFlt-1浓度显著高于未处理组(P＜0.05).Poly(I∶C)刺激原代滋养细胞后sFlt-1 mRNA水平升高,PlGF mRNA水平下降(P＜0.05).Poly(I∶C)诱导sFlt-1 mRNA的表达呈时间和剂量依赖性,24 h时效分析见其在处理2h达到峰值,PlGF mRNA则跌至最低水平(P＜0.05).Poly(I∶C)处理8～12 h,TLR3 mRNA水平亦显著升高(P＜0.05).结论 滋养细胞TLR3信号通路激活诱导sFlt-1表达,抑制PlGF表达,导致血管生成障碍,可能参与妊娠期高血压疾病的发生.     

核因子-κB“诱饵性”寡核苷酸抑制LPS诱导的肠上皮细胞TLR4、IL-8的表达

目的： 研究NF-κB decoy寡核苷酸对LPS诱导结肠癌细胞SW480后，对TLR4、IL-8表达的影响。方法：体外培养SW480细胞，用LPS(10 μg/L)刺激3 h后，用脂质体lipofectin 2000介导NF-κB decoy ODNs转染6 h，收集细胞上清液，用ELISA法检测IL-8；提取细胞mRNA，经 RT-PCR法检测TLR4 mRNA、IL-8 mRNA的表达。并设对照组、Scrambled ODNs组、lipofectin 2000组进行比较。结果：LPS刺激后TLR4 mRNA、IL-8 mRNA和IL-8的表达明显强于对照组；用NF-κB decoy寡核苷酸干预，可以明显抑制TLR4 mRNA、IL-8 mRNA和IL-8的表达。而Scrambled ODNs组和转染剂lipofectin 2000组对其没有明显影响。结论：NF-κB decoy ODNs有望成为治疗IBD的一种新型基因药品。     

TLR4基因沉默下调TLR2信号通路在RAW264.7细胞中的抗烟曲霉孢子刺激作用

目的 研究单核巨噬细胞RAW264.7受烟曲霉孢子刺激时,TLR2和TLR4信号通路发挥的作用,以及沉默TLR4基因后,对TLR2信号通路的影响.方法 利用RNAi技术将TLR4-siRNA转染RAW264.7细胞24h后给予烟曲霉孢子刺激12h,将细胞随即分为正常组(N组)、正常+烟曲霉孢子刺激组(N+Af组)、正常+TLR4-siRNA组[TLR4(RNAi)组]、正常+TLR4-siRNA+烟曲霉孢子刺激组[ TLR4(RNAi) +Af组],RT-PCR和Western blot法检测细胞受烟曲霉孢子刺激后TLR2、TLR4、MyD88 mRNA及TNF-α蛋白的表达变化.结果 (1)TLR4基因沉默前:与N组比较,N+Af组TLR2、TLR4、MyD88 mRNA及TNF-α蛋白表达量均显著升高(P＜0.05).(2)TLR4-siRNA( 100nmoL/L)转染RAW264.7细胞,沉默效率达83％.(3)TLR4基因沉默后:与N组比较,TLR4( RNAi)组TLR2、MyD88 mRNA的表达量均显著降低(P＜0.05);与N+Af组比较,TLR4( RNAi)+Af组的TLR2、MyD88 mRNA和TNF-α蛋白的表达量均显著降低(P＜0.05);与TLR4( RNAi)组比较,TLR4(RNAi)+ Af组MyD88 mRNA表达量显著升高(P＜0.05),而TLR2 mRNA及TNF-α蛋白表达量却无显著变化(P＞0.05).结论 RAW264.7细胞受烟曲霉孢子刺激时,TLR2和TLR4信号通路被激活,通过释放促炎细胞因子TNF-α发挥抗烟曲霉孢子刺激作用;当沉默TLR4基因后,TLR2信号通路不能被很好地激活来抵抗烟曲霉孢子对细胞的刺激作用,沉默TLR4基因下调了TLR2信号通路在RAW264.7细胞中的抗烟曲霉孢子刺激作用,可能TLR4较TLR2在抵抗烟曲霉孢子刺激时发挥更重要的作用.     

PolyI:C介导气道平滑肌细胞TLR3和IL-8、Eotaxin的表达

目的:观察PolyI:C对大鼠气道平滑肌细胞(ASMCs)Toll样受体3(TLR3)及炎性细胞因子表达的影响,探讨ASMCs内TLR3表达与气道炎症间的关系。方法:体外培养大鼠ASMCs,传代培养后分为正常对照组和PolyI:C刺激组,PolyI:C刺激组又分为不同的时间点。用RT-PCR法检测细胞TLR3mRNA的表达;Western blot法检测ASMCs中核因子κB(NF-κB)的蛋白含量;ELISA法检测细胞培养上清Eotaxin和IL-8的浓度。结果:与正常对照组相比较,PolyI:C刺激组ASMCs内TLR3mRNA和NF-κB蛋白表达增加(P0.05);ASMCs培养上清液中IL-8和Eotaxin浓度增加(P0.05),并存在时间依赖性。结论:PolyI:C可以上调ASMCs内TLR3的表达,活化NF-κB,增加趋化因子Eotaxin和IL-8的浓度,参与哮喘的气道炎症反应。     

Robust interferon-α and IL-12 responses by dendritic cells are related to efficient CD4+ T-cell recovery in HIV patients on ART

Amongst HIV patients with successful virological responses to antiretroviral therapy (ART), poor CD4(+) T-cell recovery is associated with low nadir CD4(+) T-cell counts and persistent immune activation. These factors might be influenced by dendritic cell (DC) function. Interferon-α-producing plasmacytoid DC and IL-12-producing myeloid DC were quantified by flow cytometry after stimulation with agonists to TLR7/8 (CL075) or TLR9 (CpG-ODN). These were compared between patients who achieved CD4(+) T-cell counts above or below 200 cells/μL after 6 months on ART (High vs. Low groups). High Group patients had more DC producing interferon-α or IL-12 at Weeks 6 and 12 on ART than Low Group patients. The frequencies of cytokine-producing DC at Week 12 were directly correlated with CD4(+) T-cell counts at baseline and at Week 12. Patients with good recovery of CD4(+) T-cells had robust TLR-mediated interferon-α responses by plasmacytoid DC and IL-12 responses by myeloid DC during early ART (1-3 months).     

Increased proportions of dendritic cells and recovery of IFNγ responses in HIV/HCV co-infected patients receiving ART

Dendritic cell (DC) numbers and functions can be affected by HIV and HCV disease, but the effects of antiretroviral therapy (ART) on DC and the implications of these changes are unclear. We examined circulating DC in samples from Indonesian patients beginning ART with advanced HIV disease and documented mild/moderate HCV hepatitis. Frequencies of myeloid and plasmacytoid DC increased after 6 months on ART, but frequencies of DC producing IL-12 or IFNα following stimulation with TLR agonists (CL075, CpG) did not change. IFNγ responses to CL075, HCV and other antigens rose over this period. Hence increased IFNγ responses during ART may be associated with increased DC frequencies rather than changes in their functional capacity.     

Adenylate cycalse toxin of Bordetella pertussis inhibits TLR-induced IRF-1 and IRF-8 activation and IL-12 production and enhances IL-10 through MAPK activation in dendritic cells

Adenylate cyclase toxin (CyaA) of Bordetella pertussis binds to CD11b/CD18 on macrophages and dendritic cells (DC) and confers virulence to the bacteria by subverting innate immune responses of the host. We have previously demonstrated that CyaA promotes the induction of IL-10-secreting regulatory T cells in vivo by modulating DC activation. Here, we examine the mechanism of immune subversion, specifically, the modulation of TLR signaling pathways in DC. We found that CyaA synergized with LPS to induce IL-10 mRNA and protein expression in DC but significantly inhibited IL-12p70 production. CyaA enhanced LPS-induced phosphorylation of p38 MAPK and ERK in DC, and inhibitors of p38 MAPK, MEK, or NF-kappaB suppressed IL-10 production in response to LPS and CyaA. However, inhibition of p38 MAPK, MEK, and NF-kappaB did not reverse the inhibitory effect of CyaA on TLR agonist-induced IL-12 production. Furthermore, CyaA suppression of IL-12 was independent of IL-10. In contrast, CyaA suppressed LPS- and IFN-gamma-induced IFN-regulatory factor-1 (IRF-1) and IRF-8 expression in DC. The modulatory effects of CyaA were dependent on adenylate cyclase activity and induction of intracellular cAMP, as an enzyme-inactive mutant of CyaA failed to modulate TLR-induced signaling in DC, whereas the effects of the wild-type toxin were mimicked by stimulation of the DC with PGE2. Our findings demonstrate that CyaA modulates TLR agonist-induced IL-10 and IL-12p70 production in DC by, respectively, enhancing MAPK phosphorylation and inhibiting IRF-1 and IRF-8 expression and that this is mediated by elevation of intercellular cAMP concentrations.     

Combined Toll-like receptor agonists synergistically increase production of inflammatory cytokines in human neonatal dendritic cells

Dendritic cells (DC) are thought to be responsible for the reduced ability of human newborns to induce protective T-helper 1 (Th1) immune responses. The key player in Th1 differentiation, interleukin-12 (IL-12), is primarily produced in response to Toll-like receptor (TLR) binding by adult DC but not by neonatal DC. The potential use of various TLR agonist combinations for initiating neonatal monocyte-derived DC to prime Th1 responses was investigated. Single TLR ligands induced maturation only in adult DC; neonatal DC matured with combined targeting of TLR3/TLR8 or TLR4/TLR8, based on the expression of maturation markers. Similarly, the synergistic effects of combined TLR ligands could also be shown with adult and neonatal cytokine production, but different expression patterns were noted. In particular, IL-12p70 was produced by neonatal DC exclusively after combined TLR stimulation. Surprisingly, it was found that supernatants of combined stimulated neonatal DC could induce interferon-gamma production in autologous na?ve T cells. Moreover, this interferon-gamma secretion was blocked by anti-IL-12p70 antibodies and increased after addition of recombined IL-12. In conclusion, these findings underline the differences between adult and neonatal DC and might suggest new strategies for promoting newborn Th1 immunity in response to pathogens and vaccine antigens.     

Neonatal goats display a stronger TH1-type cytokine response to TLR ligands than adults

In mammals, Toll-like receptors play a critical role in initiating innate immune responses and modulating adaptive immunity, by recognizing conserved microbial molecular patterns. This study was undertaken to identify specific features of the responses to synthetic toll-like receptor (TLR) agonists in goats, for the definition of tailored immunostimulation strategies. We show here, in contrast to what has been shown in mice, that mesenteric lymph nodes (MLNs) cells and splenocytes from neonatal goats produce much higher levels of TH1-type cytokines than adults in response to various TLR agonists. IL-12 was identified as a critical cytokine for IFNgamma production by CD8(+) neonatal cells. The higher level of IL-12 production by neonatal MLN and spleen cells than by adult cells was not correlated with a higher level of TLR expression or lower levels of production of the regulatory cytokine IL-10. In neonates, two cell populations-class II(+) CD8(+) and class II(+) CD8(-) cells-produce IL-12 in response to R848 and Poly I:C, respectively. Thus, goat kids have characteristics that could be exploited to favor development of the TH1-type responses critical for the control of intracellular pathogens.     

转录因子Hlx修饰树突状细胞系DC2.4的功能研究

目的:在鼠树突状细胞系DC2.4中瞬时表达Th1特异的核转录Hlx,探讨该转录因子对树突状细胞系DC2.4的功能的影响。方法:含有Hlx基因全部编码序列的真核表达载体PIRES2-EGFP/Hlx通过脂质体转染的方法转入DC细胞,转染效率应用FACS鉴定;Hlx基因在DC中表达情况应用RT-PCR和Real-time PCR检测。转染48 h后,分别从细胞因子,表面分子,吞噬功能,单向混合淋巴细胞反应方面对DC功能进行探讨。结果:真核表达载体PIRES2-EGFP/Hlx成功转入DC细胞,转染率可高达60%;在树突状细胞系DC2.4中瞬时高表达Hlx可以有效的增进成熟标志CD80,CD86,MHCII分子的表达,减弱DC的吞噬功能,IL-12表达量增加,但同时却高表达IL-10,TGF-β并表现为可抑制淋巴细胞增殖。结论:在小鼠树突状细胞系DC2.4中瞬时高表达Hlx可有效地促进DC2.4的成熟,但是在功能方面多表现为调节性DC的功能。     

IL-10-dependent partial refractoriness to Toll-like receptor stimulation modulates gut mucosal dendritic cell function

The default response of the intestinal immune system to most antigens is the induction of immunological tolerance, which is difficult to reconcile with the constant exposure to ligands for TLR and other pattern recognition receptors. We showed previously that dendritic cells (DC) from the lamina propria of normal mouse intestine may be inherently tolerogenic and here we have explored how this might relate to the expression and function of Toll-like receptors (TLR). Lamina propria (LP) DC showed higher levels of TLR 2, 3, 4 and 9 protein expression than spleen and MLN DC, with most TLR-expressing DC in the gut being CD11c(lo), class II MHC(lo), CD103(-), CD11b(-) and F4/80(-). TLR expression by lamina propria DC was low in the upper small intestine and higher in distal small intestine and colon. Freshly isolated lamina propria DC expressed some CD40, CD80, CD86 and functional CCR7. These were up-regulated on CD11c(lo), but not on CD11c(hi) LP DC by stimulation via TLR. However, there was little induction of IL-12 by either subset in response to TLR ligation. This was associated with constitutive IL-10 production and was reversed by blocking IL-10 function. Thus, IL-10 may maintain LP DC in a partially unresponsive state to TLR ligation, allowing them to have a critical role in immune homeostasis in the gut.