不同严重程度溃疡性结肠炎患者机体炎症状态和TLRs表达与肠道菌群失调的相关性

目的 探讨不同严重程度溃疡性结肠炎（UC）患者机体炎症状态和Toll样受体（TLRs）表达与肠道菌群紊乱的关系。方法 选取2021年1月至2022年1月我院诊治的102例UC患者作为研究组，以同期50例体格检查健康者作为对照组，对两组受试者的肠道菌群进行检测，同时检测患者TLR2、TLR4、TLR5、TLR9以及C反应蛋白（CRP）、白细胞介素（IL）6表达水平。指标间的关系采用Pearson相关性分析。结果 研究组患者肠道双歧杆菌、嗜酸乳杆菌、拟杆菌数量均低于对照组，大肠埃希菌、肠球菌数量均高于对照组，且研究组中活动期患者肠道双歧杆菌、嗜酸乳杆菌、拟杆菌数量低于缓解期患者，而大肠埃希菌、肠球菌数量均高于缓解期患者（均P<0.05）。研究组患者TLR2、TLR4、TLR5、TLR9相对表达量均高于对照组，且研究组中活动期患者TLRs相对表达量均高于缓解期患者（均P<0.05）。研究组患者CRP、IL-6水平均高于对照组，且研究组中活动期患者CRP、IL-6水平均高于缓解期患者（均P<0.05）。Pearson相关性分析结果显示，不同疾病严重程度UC患者机体炎症状态和T...     

血清TREM-1、TLR4及IL-37水平与变应性鼻炎患者T淋巴细胞亚群的关系研究

目的：探讨血清髓系细胞触发受体-1(TREM-1)、Toll样受体4(TLR4)及白细胞介素-37(IL-37)水平与变应性鼻炎(AR)患者T淋巴细胞亚群的关系研究。方法：选取2021年3月～2023年3月南京医科大学附属淮安第一医院和江苏省苏北人民医院收治的AR患者100例纳入AR组,另选取同期体检健康志愿者100名纳入对照组,根据病情程度将AR患者分为中重度AR组63例和轻度AR组37例。采用酶联免疫吸附法与流式细胞术检测血清TREM-1、TLR4、IL-37水平与外周血T淋巴细胞亚群。采用Pearson/Spearman分析AR患者血清TREM-1、TLR4、IL-37水平与外周血T淋巴细胞亚群的相关性。绘制受试者工作特征(ROC)曲线分析血清TREM-1、TLR4、IL-37水平对AR的诊断价值。结果：与对照组比较,AR组血清TREM-1、TLR4和外周血CD8⁺比例升高,血清IL-37和外周血CD3⁺、CD4⁺、CD4⁺/CD8⁺降低(P<0.05)。与轻度AR...     

胰腺癌患者循环肿瘤细胞及TLR4、TLR9、myd88的表达水平与其化疗效果及转移、复发的关系

目的:探讨胰腺癌患者循环肿瘤细胞(CTC)中Toll样受体4(TLR4)、Toll样受体9(TLR9)、髓样分化因子88(myd88)的表达水平与患者化疗效果及转移、复发的关系。方法:将我院2015年6月-2016年6月收治并确诊的48例胰腺癌患者作为试验组,收集患者循环肿瘤细胞(CTC),检测其TLR4、TLR9、myd88信号表达情况,探讨其TLR4、TLR9、myd88信号表达水平与患者化疗效果及转移、复发的关系。结果:48例胰腺癌患者检出CTC 35例,检出率为72.9%。胰腺癌死亡、转移、复发患者TLR4、TLR9、myd88表达水平分别高于其存活、未转移、未复发患者,组间具有统计学差异(P0.05)。胰腺癌化疗效果CR患者TLR4、TLR9、myd88表达水平显著低于其化疗效果PR、SD、PD患者,且四组间差异具有统计学意义(P0.05);TLR4、TLR9、myd88表达水平与被膜受侵犯、淋巴结转移、肿瘤大小、CA199水平呈正相关(P0.05)。结论:胰腺癌患者CTC中TLRs/myd88信号表达水平与患者化疗效果及转移、复发密切相关。     

麻杏石甘汤对风热闭肺证肺炎支原体肺炎患儿血清TLR2、TLR4和Th1/Th2免疫平衡的影响

摘要 目的：探讨麻杏石甘汤对风热闭肺证肺炎支原体肺炎（MPP）患儿血清Toll样受体（TLR）2、TLR4和辅助性T细胞（Th）1/Th2免疫平衡的影响。方法：按照随机数字表法将北京中医药大学东直门医院2021年1月到2023年1月收治的100例MPP患儿分为联合组（常规西医治疗结合麻杏石甘汤治疗，50例）和对照组（常规西医治疗，50例）。对比两组中医证候积分、潮气呼吸肺功能指标、TLR2、TLR4和Th1/Th2免疫平衡指标变化情况。结果：治疗14 d后，联合组发热恶风、微有汗出、咳嗽、口渴欲饮、呼吸急促、痰稠色黄、咽红评分低于对照组（P<0.05）。治疗14 d后，联合组潮气量（TV）、吸气时间与呼气时间之比（I/E）、达峰容积比（VPEF/VE）高于对照组，呼吸频率（RR）低于对照组（P<0.05）。治疗14 d后，联合组TLR2、TLR4低于对照组（P<0.05）。治疗14 d后，联合组干扰素γ （IFN-γ）、IFN-γ/白细胞介素-4（IL-4）低于对照组，IL-4高于对照组（P<0.05）。结论：麻杏石甘汤治疗风热闭肺证MPP患儿，可促进临床症状改善，同时还可改善患儿肺功能和免疫功能，调节血清TLR2、TLR4水平。     

HBeAg对健康人单核细胞来源树突状细胞表面TLR2和PD-L1表达水平的影响

目的:观察乙型肝炎e抗原(HBeAg)对健康人单核细胞来源的树突状细胞(Dendritic cells,DCs)表面Toll样受体2(TLR 2)和程序性死亡配体-1(PD-L1)表达的影响,进一步探讨乙肝病毒感染慢性化过程中HBeAg的作用机制.方法:利用Ficoll分离法分离健康人外周血单个核细胞,经重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、重组人白细胞介素4(rhIL-4)诱导培养树突状细胞,将第7天的细胞分成三组,分别为HBeAg刺激组、OVA无关蛋白对照组和未刺激组,经流式细胞仪检测CD11c+细胞袁面TLR2以及PD-L1的表达水平.结果:健康人单核细胞来源的树突状细胞经过HBeAg刺激9h后,CD1 1c+细胞表面TLR 2的表达水平较未刺激组以及OVA无关蛋白对照组明显下降(P＜0.01),同时可见CD11c+细胞表面PD-L1表达水平显著增加(P＜0.01).结论:HBeAg可以下调DCs表面TLR2受体的表达,并上调其表面负性调节因子PD-L1的表达.HBV可通过HBeAg作用于抗原递呈细胞相关表面受体,从而影响了其正常功能,并最终影响免疫系统对病毒的清除作用,导致乙肝病毒感染的慢性化.     

强直性脊柱炎患者外周血TNF-α、IL-17、IL-33的表达及与疾病活动程度的相关性研究

目的:探讨强直性脊柱炎(AS)患者外周血肿瘤坏死因子-α(TNF-α)、白细胞介素-17(IL-17)、白细胞介素-33(IL-33)的表达及与疾病活动程度的相关性。方法:选取2016年5月到2017年6月在我院接受治疗的AS患者74例作为研究组,另选取同期在我院体检的健康志愿者40例作为对照组,根据AS病情活动评分量表(BASDAI)将研究组患者分为A组(BASDAI4分,32例)和B组(BASDAI得分≦4分,42例),比较研究组和对照组外周血中TNF-α、IL-17、IL-33水平,比较A组和B组外周血中TNF-α、IL-17、IL-33、C反应蛋白(CRP)、红细胞沉降率(ESR),采用Spearman相关性分析TNF-α、IL-17、IL-33水平与BASDAI得分及CRP、ESR的相关性。结果:研究组外周血中TNF-α、IL-17、IL-33水平明显高于对照组,差异有统计学意义(P0.05),A组外周血中TNF-α、IL-17、IL-33、CRP、ESR明显高于B组,差异有统计学意义(P0.05);AS患者TNF-α、IL-17、IL-33水平与BASDAI得分及CRP、ESR水平均呈正相关(P0.05)。结论:TNF-α、IL-17、IL-33在AS患者外周血中呈现高表达,并且与患者BASDAI得分及CRP、ESR水平呈正相关。     

TLR4mAb 对急性溃疡性结肠炎模型大鼠TLR4、TNF-alpha、IL-1beta 表达的影响

目的：探讨Toll 样受体4 单克隆抗体（TLR4mA）对溃疡性结肠炎（UC）模型大鼠肠TLR4、TNF-alpha、IL-1beta表达的影响及其对 UC 的可能作用机制。方法：30只SD 大鼠随机分为正常对照组、模型组、TLR4mAb 干预组。模型组及TLR4mAb 干预组采用三硝 基苯磺酸（TNBs）法造模,TLR4mAb 干预组给予TLR4mAb10 ug 腹腔注射,正常对照组及模型组以生理盐水代替TLR4mAb 腹 腔注射,剂量及频次相同,第8 天处死全部大鼠。分别进行疾病活动指数（DAI）评分及组织病理学（HPS）评分,采用免疫组化法检 测结肠组织TLR4 的原位表达,酶联免疫吸附试验（ELISA 法）检测血清肿瘤坏死因子-alpha（TNF-alpha）、白细胞介素-1beta（IL-1beta）的浓 度。结果：模型组DAI及HPS 评分均明显高于正常对照组（P<0.05）,TLR4mAb 干预组较模型组有所缓解（P<0.05）。TLR4、TNF- alpha、IL-1beta在模型组表达均明显高于正常对照组,差异有统计学意义（P<0.05）,在TLR4mAb 干预组的表达均明显低于模型组,差异 有统计学意义（P<0.05）。结论：急性溃疡性结肠炎的发病与异常免疫反应有关。TLR4mAb 可影响肠黏膜TLR4 及下游炎症因子 TNF-alpha、IL-1beta的表达,减轻急性溃疡性结肠炎大鼠的肠道炎症反应。     

TLR4mAb对急性溃疡性结肠炎模型大鼠TLR4、TNF-α、IL-1β表达的影响

目的:探讨Toll样受体4单克隆抗体(TLR4m A)对溃疡性结肠炎(UC)模型大鼠肠TLR4、TNF-α、IL-1β表达的影响及其对UC的可能作用机制。方法:30只SD大鼠随机分为正常对照组、模型组、TLR4m Ab干预组。模型组及TLR4m Ab干预组采用三硝基苯磺酸(TNBs)法造模,TLR4m Ab干预组给予TLR4m Ab10μg腹腔注射,正常对照组及模型组以生理盐水代替TLR4m Ab腹腔注射,剂量及频次相同,第8天处死全部大鼠。分别进行疾病活动指数(DAI)评分及组织病理学(HPS)评分,采用免疫组化法检测结肠组织TLR4的原位表达,酶联免疫吸附试验(ELISA法)检测血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)的浓度。结果:模型组DAI及HPS评分均明显高于正常对照组(P0.05),TLR4m Ab干预组较模型组有所缓解(P0.05)。TLR4、TNF-α、IL-1β在模型组表达均明显高于正常对照组,差异有统计学意义(P0.05),在TLR4m Ab干预组的表达均明显低于模型组,差异有统计学意义(P0.05)。结论:急性溃疡性结肠炎的发病与异常免疫反应有关。TLR4m Ab可影响肠黏膜TLR4及下游炎症因子TNF-α、IL-1β的表达,减轻急性溃疡性结肠炎大鼠的肠道炎症反应。     

有氧运动抑制自发性高血压大鼠心肌中HMGB1/TLR4炎症通路

目的:探讨有氧运动对自发性高血压大鼠心肌中高迁移率族蛋白1(high mobility group box-1 protein, HMGB1)/Toll样受体4(toll like receptor 4,TLR4)炎症通路的影响。方法:成年雄性大鼠分为三组:同源正常血压大鼠(Wistar-Kyoto rats,WKY),自发性高血压大鼠(spontaneoully hypertensive rat, SHR),自发性高血压大鼠运动组(SHR+exercise,SHR+EX)。运动为中等强度跑台运动,自制跑台,倾斜角设置为10度,运动强度逐步增强,持续12周。尾动脉检测血压,收集左心室,Masson染色检测心肌组织纤维化水平,RT-PCR法检测心肌中纤维化相关基因的m RNA水平,ELISA法检测炎症因子白介素6(Interleukin 6, IL-6)和肿瘤坏死因子α(tumor necrosis factor alpha, TNF-α)的蛋白水平,Western blot法检测HMGB1和TLR4的蛋白表达。结果:有氧运动可以明显降低SHR大鼠心肌中HMGB1(P0.05)和TLR4(P0.05)的蛋白表达以及炎症因子IL-6(P0.05)和TNF-α(P0.05)的蛋白水平。经过的有氧运动之后,SHR大鼠的体重(P0.05)、收缩压(P0.05)、舒张压明显降低(P0.05);心肌纤维化水平和心肌中I型胶原(P0.05)、转化生长因子-β(transforming growth factor-β,TGF-β,P0.05)、α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA,P0.05)的m RNA水平出现显著下降。结论:有氧运动可抑制自发性高血压大鼠心肌中HMGB1/TLR4炎症通路。     

孟鲁司特治疗儿童过敏性紫癜的临床疗效及对Toll样受体和IL-6, IL-8 表达的影响

目的:研究孟鲁司特治疗儿童过敏性紫癜的临床疗效及对Toll样受体(TLRs)和白介素-6(IL-6)、白介素-8(IL-8)表达的影响。方法:选取2011年2月到2015年2月我院收治的儿童过敏性紫癜患者150例,按照随机数字表法将患者分为研究组和对照组,每组75例,对照组给予常规治疗,研究组在对照组的基础上给予孟鲁司特,应用荧光定量PCR技术测量TLR2、TLR5和TLR9mRNA基因表达情况,应用酶联免疫吸附(EILSA)法测定患者血清中IL-6和IL-8水平,并比较两组临床疗效,皮疹消退、关节肿痛以及腹痛缓解时间。结果:研究组总有效率97.3%(73/75)显著高于对照组81.3%(61/75),两组比较差异具有统计学意义(P0.05);研究组皮疹消退、关节肿痛以及腹痛缓解时间均显著短于对照组,两组比较差异具有统计学意义(P0.05);两组治疗后TLR2、TLR5和TLR9m RNA表达水平显著低于治疗前,且研究组显著低于对照组,比较差异具有统计学意义(P0.05);两组治疗后IL-6和IL-8水平显著低于治疗前,且研究组显著低于对照组,比较差异具有统计学意义(P0.05)。结论:孟鲁司特治疗儿童过敏性紫癜具有较好的临床疗效,能显著降低TLRs、IL-6和IL-8水平。     

Cigarette smoke increases TLR4 and TLR9 expression and induces cytokine production from CD8+ T cells in chronic obstructive pulmonary disease

Background

Cigarette smoke is a major risk factor for chronic obstructive pulmonary disease (COPD), an inflammatory lung disorder. COPD is characterized by an increase in CD8⁺ T cells within the central and peripheral airways. We hypothesized that the CD8⁺ T cells in COPD patients have increased Toll-like receptor (TLR) expression compared to control subjects due to the exposure of cigarette smoke in the airways.

Methods

Endobronchial biopsies and peripheral blood were obtained from COPD patients and control subjects. TLR4 and TLR9 expression was assessed by immunostaining of lung tissue and flow cytometry of the peripheral blood. CD8⁺ T cells isolated from peripheral blood were treated with or without cigarette smoke condensate (CSC) as well as TLR4 and TLR9 inhibitors. PCR and western blotting were used to determine TLR4 and TLR9 expression, while cytokine secretion from these cells was detected using electrochemiluminescence technology.

Results

No difference was observed in the overall expression of TLR4 and TLR9 in the lung tissue and peripheral blood of COPD patients compared to control subjects. However, COPD patients had increased TLR4 and TLR9 expression on lung CD8⁺ T cells. Exposure of CD8⁺ T cells to CSC resulted in an increase of TLR4 and TLR9 protein expression. CSC exposure also caused the activation of CD8⁺ T cells, resulting in the production of IL-1β, IL-6, IL-10, IL-12p70, TNFα and IFNγ. Furthermore, inhibition of TLR4 or TLR9 significantly attenuated the production of TNFα and IL-10.

Conclusions

Our results demonstrate increased expression of TLR4 and TLR9 on lung CD8⁺ T cells in COPD. CD8⁺ T cells exposed to CSC increased TLR4 and TLR9 levels and increased cytokine production. These results provide a new perspective on the role of CD8⁺ T cells in COPD.     

Regulation of TLR2 Expression and Function in Human Airway Epithelial Cells

Toll-like receptor (TLR1–6) mRNAs are expressed in normal human bronchial epithelial cells with higher basal levels of TLR3. TLR2 mRNA and plasma membrane protein expression was enhanced by pretreatment with Poly IC, a synthetic double-stranded RNA (dsRNA) known to activate TLR3. Poly IC also enhanced mRNA expression of adaptor molecules (MyD88 and TIRAP) and coreceptors (Dectin-1 and CD14) involved in TLR2 signaling. Additionally, mRNA expression of TLR3 and dsRNA-sensing proteins MDA5 and RIG-I increased following Poly IC treatment. In contrast, basal mRNA expression of TLR5 and TLR2 coreceptor CD36 was reduced by 77% and 62%, respectively. ELISA of apical and basolateral solutions from Poly IC-stimulated monolayers revealed significantly higher levels of IL-6 and GM-CSF compared with the TLR2 ligand PAM₃CSK₄. Pretreatment with anti-TLR2 blocking antibody inhibited the PAM₃CSK₄-induced increase in IL-6 secretion after Poly IC exposure. An increase in IL-6 secretion was also observed in cells stimulated with Alternaria extract after pretreatment with Poly IC. However, IL-6 secretion was not stimulated by zymosan or lipothechoic acid (LTA). These data demonstrated that upregulation of TLR2 following exposure to dsRNA enhances functional responses of the airway epithelium to certain (PAM₃CSK₄), but not all (zymosan, LTA) TLR2 ligands and that this is likely due to differences in coreceptor expression.     

The biological effects of CRP are not attributable to endotoxin contamination: evidence from TLR4 knockdown human aortic endothelial cells

C-reactive protein (CRP) is the prototypic marker of inflammation and a strong predictor of cardiovascular events in humans. There are questions regarding the validity of the biological effects reported for CRP, in spite of adherence to rigorous control measures minimizing endotoxin [lipopolysaccharide (LPS)] contamination in these in vitro studies. In this study, we addressed the key question of endotoxin contamination in CRP preparations using Toll-like receptor 4 (TLR4) knockdown endothelial cells. Human aortic endothelial cells (HAECs) transfected with prevalidated TLR4 small interfering RNA (siRNA) and scrambled siRNA controls were challenged with pleural fluid-derived CRP or LPS for 12-16 h. Secreted interleukin-6 (IL-6), IL-1beta, IL-8, and plasminogen activator inhibitor-1 (PAI-1) levels and endothelial Nitric oxide synthase (eNOS) activity were determined. TLR4 knockdown in HAECs significantly decreased LPS-induced IL-1beta, IL-6, and IL-8, whereas the stimulatory effects of CRP were similar in both scrambled control and TLR4 knockdown cells. Furthermore, CRP significantly stimulated PAI-1 levels in both control and TLR4-transfected cells and inhibited eNOS activity, whereas LPS effects were negated in TLR4-transfected cells. The data presented cogently demonstrate and further confirm that the biological effects of CRP on HAECs are independent of LPS and thus are attributable to native protein per se. This is the first study to positively authenticate the significance of earlier in vitro reports on CRP biological effects.     

TLR9 agonists induced cell death in Burkitt's lymphoma cells is variable and influenced by TLR9 polymorphism

Toll-like receptor 9 (TLR9) triggering is a promising novel strategy to combat cancer as it induces innate and adaptive immunity responses. B-cell lymphoma is unique in this context as tumor cells express TLR9 and may harbor latent Epstein-Barr virus (EBV), a gamma-herpesvirus with remarkable oncogenic potential when latent. Latent EBV may be promoted by TLR9 triggering via suppression of lytic EBV. Here, we elaborated an initial assessment of the impact of TLR9 triggering on EBV-positive and EBV-negative B-cell lymphoma using Burkitt''s lymphoma (BL) cell lines as an in vitro model. We show that, independent of the presence of EBV, the TLR9 ligand oligodeoxynucleotide (ODN) CpG-2006 may or may not induce caspase-dependent cell death in BL cells. Moreover, ODN CpG-2006-induced cell death responses of BL cells were associated with TLR9 single-nucleotide polymorphisms (SNPs) rs5743836 or rs352140, which we detected in primary BL tumors and in peripheral blood from healthy individuals at similar frequencies. Thus, our findings suggest that the effect of TLR9 agonists on BL cells should be tested in vitro before installment of therapy and TLR9 SNPs in BL patients should be determined as potential biological markers for the therapeutic response to treatment targeting innate immunity.     

Toll-like receptor (TLR) 2-9 agonists-induced cytokines and chemokines: I. Comparison with T cell receptor-induced responses

The cells of innate and adaptive immunity, although activated by different ligands, engage in cross talk to ensure a successful immune outcome. To better understand this interaction, we examined the demographic picture of individual TLR (TLRs 2-9) -driven profiles of eleven cytokines (IFN-alpha/beta, IFN-gamma, IL-12p40/IL-12p70, IL-4, 1L-13, TNF-alpha, IL-1beta, IL-2, IL-10) and four chemokines (MCP-1, MIP1beta, IL-8, and RANTES), and compared them with direct T-cell receptor triggered responses in an assay platform using human PBMCs. We find that T-cell activation by a combination of anti-CD3/anti-CD28/PHA induced a dominant IL-2, IL-13, and Type-II interferon (IFN-gamma) response without major IL-12 and little Type-I interferon (IFN-alphabeta) release. In contrast, TLR7 and TLR9 agonists induced high levels of Type-I interferons. The highest IFN-gamma levels were displayed by TLR8 and TLR7/8 agonists, which also induced the highest levels of pro-inflammatory cytokines IL-12, TNF-alpha, and IL-1beta. Amongst endosomal TLRs, TLR7 displayed a unique profile producing weak IL-12, IFN-gamma, TNF-alpha, IL-1beta, and IL-8. TLR7 and TLR9 resembled each other in their cytokine profile but differed in MIP-1beta and MCP1 chemokine profiles. Gram positive (TLR2, TLR2/6) and gram negative (TLR4) pathogen-derived TLR agonists displayed significant similarities in profile, but not in potency. TLR5 and TLR2/6 agonists paralleled TLR2 and TLR4 in generating pro-inflammatory chemokines MCP-1, MIP-1beta, RANTES, and IL-8 but yielded weak TNF-alpha and IL-1 responses. Taken together, the data show that diverse TLR agonists, despite their operation through common pathways induce distinct cytokine/chemokine profiles that in turn have little or no overlap with TCR-mediated response.     

乳酸杆菌脂磷壁酸抑制脂多糖诱导的大鼠肝脏Kupffer 细胞TLR4 通路 的激活

目的：探讨保加利亚乳酸杆菌脂磷壁酸（Lipoteichoic Acid of ,LBG-LTA）对大鼠肝脏Kupffer细胞 Toll样受体4（Toll-like receptor 4,TLR4）信号通路的作用。方法：雄性健康Wistar大鼠10 只（2 月龄,体重250～300 g）处死后,分 离培养肝脏Kupffer 细胞;培养LBG,并提取制备LBG-LTA;Kupffer 细胞,在有或无LBG-LTA（0.1、1、10 ug/mL）预处理的情况 下,给予脂多糖（lipopolysaccharide,LPS,1 EU/mL）刺激后,Western blot 检测各孔Kupffer细胞的TLR4、TANK 结合激酶1（TANK binding kinase-1,TBK1）及核中的核因子B（nuclear factor-kB,NF-kB）水平,酶联免疫吸附法检测各孔培养上清中的肿瘤坏死因子 alpha（tumor necrosis factor-alpha,TNF-alpha）和白介素1beta（interleukin-1beta,IL-1beta）。结果：分离的Kupffer 细胞经不同浓度LBG-LTA 预处理 后,其在LPS刺激下所表达的TLR4、TBK1、核中NF- kB的水平及生成的TNF-alpha和IL-1茁明显低于无LBG-LTA预处理情况下的 LPS 孔（P<0.05）,且LBG-LTA 的作用呈浓度依赖性。结论：LBG-LTA以浓度依赖的方式抑制了LPS 诱导下大鼠Kupffer细胞 TLR4 通路的激活。     

Novel TLR4-antagonizing peptides inhibit LPS-induced release of inflammatory mediators by monocytes

Toll-like receptor 4 (TLR4) has become a new target for combating Gram-negative bacterium-induced sepsis. In this study, we screened peptides that can interact with TLR4 from a random 16-peptide library using yeast two-hybrid system and performed functional identification for the obtained peptides. We got two positive clones out of 1.28x10(7) transformants. The peptides were sequenced and synthesized. Protein sequence comparison confirmed that the two peptides had no homologous proteins. The two peptides were found to significantly inhibit LPS-induced NF-kappaB activation in HEK-293 cells that were transfected with TLR4 cDNA, LPS-induced IkappaBalpha (IkappaB alpha) phosphorylation and NF-kappaB activation in monocytes, and release of IL-1, IL-6, and TNF-alpha by monocytes. We further confirmed that the No. 9 peptide could bind to TLR4 extracellular domain, but the No. 24 peptide could not, suggesting that two novel peptides were identified as the antagonists of TLR4, which significantly inhibited the effects of endotoxin in vitro. The No. 9 peptide may function through binding to TLR4 extracellular domain. Our findings suggest a promising countermeasure against Gram-negative bacterium-induced sepsis.     

Acquisition of Adult-Like TLR4 and TLR9 Responses during the First Year of Life

Background

Characteristics of the human neonatal immune system are thought to be responsible for heightened susceptibility to infectious pathogens and poor responses to vaccine antigens. Using cord blood as a source of immune cells, many reports indicate that the response of neonatal monocytes and dendritic cells (DC) to Toll-like receptor (TLR) agonists differs significantly from that of adult cells. Herein, we analyzed the evolution of these responses within the first year of life.

Methodology/Principal Findings

Blood samples from children (0, 3, 6, 9, 12 month old) and healthy adults were stimulated ex vivo with bacterial lipopolysaccharide (LPS, TLR4 agonist) or CpG oligonucleotides (TLR9 agonist). We determined phenotypic maturation of monocytes, myeloid (m) and plasmacytoid (p) DC and production of cytokines in the culture supernatants. We observed that surface expression of CD80 and HLA-DR reaches adult levels within the first 3 months of life for mDCs and 6–9 months of life for monocytes and pDCs. In response to LPS, production of TNF-α, IP-10 and IL-12p70 reached adult levels between 6–9 months of life. In response to CpG stimulation, production of type I IFN-dependent chemokines (IP-10 and CXCL9) gradually increased with age but was still limited in 1-year old infants as compared to adult controls. Finally, cord blood samples stimulated with CpG ODN produced large amounts of IL-6, IL-8, IL-1β and IL-10, a situation that was not observed for 3 month-old infants.

Conclusions

The first year of life represents a critical period during which adult-like levels of TLR responses are reached for most but not all cytokine responses.     

TLR7/TLR8 Activation Restores Defective Cytokine Secretion by Myeloid Dendritic Cells but Not by Plasmacytoid Dendritic Cells in HIV-Infected Pregnant Women and Newborns

Mother-to-child transmission (MTCT) of HIV-1 has been significantly reduced with the use of antiretroviral therapies, resulting in an increased number of HIV-exposed uninfected infants. The consequences of HIV infection on the innate immune system of both mother-newborn are not well understood. In this study, we analyzed peripheral blood and umbilical cord blood (CB) collected from HIV-1-infected and uninfected pregnant women. We measured TNF-α, IL-10 and IFN-α secretion after the stimulation of the cells with agonists of both extracellular Toll-like receptors (TLRs) (TLR2, TLR4 and TLR5) and intracellular TLRs (TLR7, TLR7/8 and TLR9). Moreover, as an indicator of the innate immune response, we evaluated the responsiveness of myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) to TLRs that are associated with the antiviral response. Our results showed that peripheral blood mononuclear cells (PBMCs) from HIV-1-infected mothers and CB were defective in TNF-α production after activation by TLR2, TLR5, TLR3 and TLR7. However, the TNF-α response was preserved after TLR7/8 (CL097) stimulation, mainly in the neonatal cells. Furthermore, only CL097 activation was able to induce IL-10 and IFN-α secretion in both maternal and CB cells in the infected group. An increase in IFN-α secretion was observed in CL097-treated CB from HIV-infected mothers compared with control mothers. The effectiveness of CL097 stimulation was confirmed by observation of similar mRNA levels of interferon regulatory factor-7 (IRF-7), IFN-α and TNF-α in PBMCs of both groups. The function of both mDCs and pDCs was markedly compromised in the HIV-infected group, and although TLR7/TLR8 activation overcame the impairment in TNF-α secretion by mDCs, such stimulation was unable to reverse the dysfunctional type I IFN response by pDCs in the HIV-infected samples. Our findings highlight the dysfunction of innate immunity in HIV-infected mother-newborn pairs. The activation of the TLR7/8 pathway could function as an adjuvant to improve maternal-neonatal innate immunity.