Toll样受体及其信号转导

Toll样受体(TLR)介导着绝大部分哺乳动物、昆虫及植物的宿主防御. TLR4与配体结合涉及膜抗原CD14和分泌蛋白MD-2的调节并一起形成受体复合物, 然后与接头分子MyD88结合, 使IRAK磷酸化, 再使TRAF6寡聚化, 随后激活控制着各种效应基因表达的转录因子NF-κB.     

绝经后骨质疏松症TNF-α通过激活NF-κB促进RANKL诱导的破骨细胞形成

本研究检测了绝经后骨质疏松症妇女的肿瘤坏死因子-α(TNF-α)和雌激素水平,并探讨了TNF-α对破骨前体细胞RAW264.7中破骨细胞标志物核因子κB受体激活因子(nuclear factor kappa-B, RANK)、组织蛋白酶K (Cathepsin K, CTSK)和凝血酶受体激活肽(thrombin receptor activating peptide, TRAP)以及核因子-κB (NF-κB)亚基(p65)和NF-κB抑制蛋白(IκBα)的影响。研究结果表明,绝经后骨质疏松症患者的TNF-α水平显著升高,而雌二醇水平显著降低。核因子κB受体激活因子配体(receptor activator for NF-κBligand, RANKL)处理1周后,破骨前体细胞RAW264.7中破骨细胞标志物RANK、CTSK和TRAP的mRNA和蛋白高度表达。与RANKL对照组相比,TNF-α处理可上调RANK、CTSK和TRAP m RNA的表达。但是,仅TNF-α不能诱导培养的RAW264.7细胞分化为破骨细胞成。TNF-α以剂量依赖性方式诱导NF-κB亚基p65和IκBα磷酸化,而NF-κB抑制剂处理则有效降低了RANK和TRAP的表达。本研究结论表明,绝经后骨质疏松症中TNF-α通过激活NF-κB来促进RANKL诱导的破骨细胞形成。     

NF-κB在LPS诱导的帕金森病模型大鼠中的作用

目的:研究NF-κB在脂多糖(LPS)诱导的帕金森病(PD)模型大鼠中的作用.方法:52只SD大鼠按体重随机分为3组,实验组(LPS组)、预防组(PDTC+LPS组)和对照组,每组14只:(1)实验组SD大鼠按体重50 mg/kg经腹腔注射生理盐水后经脑立体定位单侧黑质注射LPS4μl/只(4μg/只)后7 d制成帕金森病模型;(2)预防组SD大鼠按体重50 mg/kg经腹腔注射PDTC lh后再经脑立体定位单侧黑质注射LPS 4μl/只(4μg/只);(3)对照组SD大鼠按体重50 mg/kg经腹腔注射生理盐水后经脑立体定位单侧黑质注射生理盐水4μl/只.7 d后观察三组大鼠皮下注射阿扑吗啡后的行为学变化,采用免疫荧光法检测注射7 d后黑质部NF-κBp65的表达,并用western blotting检测其蛋白表达.结果:7d后,实验组大鼠有明显的行为学变化,预防组和对照组则无异常行为学表现.免疫荧光结果显示,与注射对侧相比,实验组大鼠经脑立体定位注射内毒素LPS后,注射侧黑质部TH表达显著减少,而NF-κBp65阳性表达则显著增加,western blotting得到同样结果(P＜0.05);预防组和对照组大鼠黑质部TH和NF-κBp65阳性表达无显著性变化,western blotting得到同样结果(P＞0.05).结论:NF-κB参与了LPS PD大鼠发病过程,黑质中NF-κBp65过度激活是LPS对大鼠造成损害作用的机制之一.预先使用PDTC对LPS PD鼠起到一定神经保护作用.     

miR-21对心肌缺血大鼠心肌细胞TLR-4/NF-κB通路的影响

摘要 目的：探究微小核糖核酸-21（miR-21）对心肌缺血大鼠心肌细胞Toll样受体4（TLR4）/核因子-κB（NF-κB）通路的影响。方法：取40只SD大鼠随机分为假手术组（10只）和建模组（30只）,建模组大鼠通过左冠状动脉前降支（LAD）结扎术建立心肌缺血大鼠模型,假手术组大鼠仅开胸后不做其他处理即缝合。将建模成功大鼠随机分为对照组（转染miR-NC慢病毒）、沉默组（转染miR-21 antagomir慢病毒）、过表达组（转染miR-21 mimics慢病毒）,假手术组注射等量生理盐水。苏木素-伊红（HE）染色观察心肌组织损伤情况;原位末端标记法（TUNEL）检测细胞凋亡率;实时定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法（WB）检测TLR-4、NF-κB、半胱氨酸天冬氨酸蛋白酶3（Caspase3）、B淋巴细胞瘤-2基因（Bcl-2）、Bcl-2关联X蛋白（Bax）信使核糖核酸（mRNA）和蛋白表达及磷酸化NF-κB p65（p-NF-κB p65）水平。结果：对照组大鼠心肌纤维排列紊乱,大量心肌细胞肿胀坏死并伴随炎症细胞浸润,沉默组大鼠心肌组织损伤情况进一步加重,而过表达组大鼠心肌组织损伤现象得到明显改善。与假手术组比,对照组、沉默组、过表达组大鼠心肌细胞凋亡率,TLR-4、NF-κB、Caspase-3、Bax mRNA与其蛋白表达,以及p-NF-κB p65水平升高,Bcl-2 mRNA及其蛋白表达降低（P＜0.05）;与对照组比,沉默组大鼠心肌细胞凋亡率,TLR-4、NF-κB、Caspase-3、Bax mRNA表达与其蛋白表达,以及p-NF-κB p65水平升高,Bcl-2 mRNA及其蛋白表达降低（P＜0.05）,过表达组大鼠心肌细胞凋亡率,TLR-4、NF-κB、Caspase-3、Bax mRNA与其蛋白表达,以及p-NF-κB p65水平降低,Bcl-2 mRNA及其蛋白表达升高（P＜0.05）。结论：下调miR-21表达可促进TLR-4/NF-κB通路表达,加重心肌缺血大鼠心肌损伤。     

Toll样受体4表达的调节

Toll样受体4(TLR4)是最早发现的人类Toll样受体,主要表达于树突状细胞和巨噬细胞等免疫细胞表面,在免疫应答和炎症反应中起重要作用。本文综述了影响TLR4表达的包括上调因素和下调因素在内的一些主要调节因素。     

TRAF6 与NF-κB 在特发性炎症性肌病小鼠肌肉中的表达及意义

目的:研究肿瘤坏死因子受体相关因子6(TRAF6)与核转录因子κB(NF-κB)在特发性炎症性肌病(IIMs)中的表达情况,探讨TRAF6在IIMs发病中的作用及机制。方法:30只雌性BALB/c小鼠随机分为5组(每组6只),A:正常对照组;B~E:IIMs模型自第一次免疫后分别在1周、2周、3周、4周末处理组;采用实时荧光定量PCR方法检测各组小鼠肌肉组织中TRAF6与NF-κB m RNA表达水平。结果:(1)IIMs各组小鼠肌肉中TRAF6与NF-κB m RNA与正常对照组相比表达均有不同程度升高(P0.01),第2周末时升高最为显著(P0.01),第3周、4周呈下降趋势(P0.01);(2)IIMs小鼠各组肌肉组织中TRAF6与NF-κB m RNA表达水平与肌肉炎症程度呈正相关(r=0.940,r=0.908,P0.01),前二者之间也呈显著正相关(r=0.944,P0.01)。结论:TRAF6、NF-κB m RNA表达在IIMs小鼠肌肉中上调,TRAF6可能通过NF-κB的激活在IIMs发生发展过程中发挥重要作用。     

乳酸调控大鼠肠黏膜微血管内皮细胞的NF-κB信号通路

目的探讨内毒素（LPS）刺激大鼠肠黏膜微血管内皮细胞（RIMMVECs）后,乳酸（LA）调控NF-κB信号通路中磷酸化IκBα和NF-κB p65蛋白表达情况,肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）mRNA表达情况,阐明乳酸发挥作用的最佳时间及其调控NF-κB信号通路的部位。方法提取RIMMVECs总蛋白和总RNA,用Western blotting检测NF-κB p65、IκBα及p-IκBα蛋白表达水平,用real-time PCR对TNF-α和IL-6 mRNA进行定量检测。结果乳酸能降低LPS诱导RIMMVECs分泌的TNF-α和IL-6 mRNA表达水平,并分别于24 h和3 h下调效果最明显;乳酸能抑制IκBα磷酸化及NF-κB转录活性,并于4～8 h达到最佳效果;乳酸发挥作用部位是抑制信号通路中IκBα磷酸化。结论乳酸通过抑制IκBα磷酸化而阻断NF-κB的激活,抑制下游炎性因子表达,进而发挥出很好的预防炎症效果。     

Toll样受体4信号转导通路的干预

Toll样受体4(Toll like receptor 4,TLR4)是广泛表达于哺乳动物的跨膜受体,由于TLR4在人体的高表达与各种炎症反应相关联,抑制过高的TLR4表达可能是控制机体炎症损伤的新途径.目前的研究主要是针对TLR4的直接阻断与对TLR4的信号转导通路的抑制.由于TLR4的信号转导通路已经较为明确,从而研究对TLR4信号转导通路的抑制可能会对机体过强的炎症反应及损伤的控制产生有益作用.本文就当前针对抑制TLR4信号转导通路的研究作一综述.     

肿瘤坏死因子α通过激活NF-κB信号通路加快肝细胞周期进程

在慢性炎症部位有易发肿瘤的倾向,大约有20%的恶性肿瘤发生与慢性炎症相关,肝细胞癌是世界第三大癌症死亡病因,其患者多数有慢性炎症病史,当炎症慢性迁延,肝细胞癌发生率明显增加.但慢性炎症与肿瘤发生与发展的细胞和分子机制仍然不清楚.利用人肝细胞株L-02细胞,研究肿瘤坏死因子α(TNF-α)对细胞周期的影响及其机制,并探讨核因子κB(NF-κB)和ERK1/2活化对细胞周期的影响,以期能更确切地阐明炎症介质TNF-α在肝细胞癌发生发展中的作用.发现TNF-α能促进肝细胞从G0/G1期向S期转换.蛋白质印迹检测表明,TNF-α能以剂量依赖方式诱导cyclinD1表达,而对cyclinE的表达无明显影响.同时TNF-α能激活NF-κB,ERK1/2,抑制NF-κB活化降低了TNF-α诱导的cyclinD1表达,导致细胞周期阻滞于G0/G1期.抑制ERK1/2活化则对细胞周期和cyclinD1表达无显著影响.结果提示,TNF-α通过活化NF-κB信号通路,诱导cyclinD1表达,加快细胞周期进程,这可能是促进肿瘤的发生发展重要机制.针对TNF-α和NF-κB的治疗可能延长慢性炎症相关性肿瘤的潜伏期和抑制肿瘤的发展.     

Toll样受体4(TLR4)基因剔除小鼠构建及初步表型分析

目的 利用CRISPR/Cas9技术构建Toll样受体4(TLR4)基因敲除小鼠模型,并观察突变小鼠对革兰氏阴性细菌脂多糖(LPS)刺激响应的变化。方法 针对TLR4基因外显子2设计并合成1对sgRNA片段,与编码Cas9的mRNA混合后通过受精卵显微注射方法,建立TLR4基因敲除小鼠,通过繁育获得基因敲除纯合子小鼠(TLR4^-/-小鼠);通过LPS刺激,分析TLR4^-/-小鼠对炎症应激的反应情况,并在分子和病理水平上和野生型对照(WT)进行比较。结果 PCR及测序检测表明TLR4基因外显子2在小鼠基因中被成功敲除;给予LPS刺激后,IL1β、IL6、MyD88、iNOS及TNFa等炎症因子的表达在野生型小鼠的心、肝和肺组织中显著上调,而在TLR4^-/-小鼠中则几乎没有变化;血生化指标显示LPS刺激后WT小鼠血清中的尿素(Urea)和肌酐(Cre)水平显著升高,而TLR4^-/-小鼠刺激前后无显著变化,病理分析同样发现TLR4^-/-小鼠能够抵抗LPS对肾组织的损伤。结论 利用CRISPR/Cas9技术成功构建了TLR4基因剔除小鼠模型,TLR4的缺失能够降低IL1β、IL6、MyD88、iNOS及TNFa炎症因子对LPS刺激的响应,抑制LPS引起的炎症反应及对组织的损伤。     

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.     

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications ¹. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages ², starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A ³ in combination with several growth factors ^4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine ⁸. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation ⁹. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation ^10,11.The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.     

ERK5 Protein Promotes, whereas MEK1 Protein Differentially Regulates, the Toll-like Receptor 2 Protein-dependent Activation of Human Endothelial Cells and Monocytes

Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation up-regulates the expression of inflammatory mediators and of TLR2 itself and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in EC and tested the hypothesis that TLR2 signaling differs in EC and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in human umbilical vein endothelial cells, primary human lung microvascular EC, and human monocytes. Additionally, we observed that, although MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the up-regulation of intracellularly expressed TLR2 and inflammatory mediators via NF-κB, JNK, and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5, and NF-κB promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in EC and monocytes and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes.     

胞浆型磷脂酶A2介导弱氧化修饰低密度脂蛋白诱导的人脐静脉内皮细胞凋亡

探讨弱氧化修饰低密度脂蛋白(MM-LDL)能否诱导人脐静脉内皮细胞(HUVECs)凋亡以及胞浆型磷脂酶A2(cPLA2)在此过程中的作用.MTT法测定细胞存活率;相差显微镜、荧光显微镜和流式细胞仪检测细胞凋亡;3H-花生四烯酸(3H-AA)预标法测定PLA2活性;蛋白质印迹检测cPLA2磷酸化;激光共聚焦显微镜检测单个细胞内钙离子浓度的变化.结果表明,MM-LDL(100～300 mg/L)作用后的HUVECs呈现凋亡典型的形态特征,凋亡率随MM-LDL浓度的增加而上升.MM-LDL能引起胞内钙离子浓度增加,cPLA2的活化及磷酸化.15 μmol/L AACOCF3和5 mmol/L EGTA在抑制cPLA2活性的同时,部分抑制MM-LDL诱导的HUVECs凋亡.加入外源性AA(50 μmol/L)能逆转AACOCF3引起的凋亡抑制.结果提示,cPLA2参与了MM-LDL诱导HUVECs凋亡的信号传递.     

Characterization of the Toll-like Receptor Expression Profile in Human Multiple Myeloma Cells

Expression and function of Toll-like receptors (TLRs) in multiple myeloma (MM) has recently become the focus of several studies. Knowledge of expression and biology of these receptors in MM will provide us with a new insight into the role of an inflammatory environment in disease progression or pathogenesis of MM. However, to date a quite heterogeneous expression pattern of TLRs in MM particularly at gene level has been described while information on the TLR expression at the protein level is largely unavailable. In this study, we investigated the TLR expression in human myeloma cell lines (HMCLs) Fravel, L363, UM6, UM9, OPM1, OPM2, U266, RPMI 8226, XG1, and NCI H929 and primary cells from MM patients at both mRNA and protein level (western blot and flow cytometry). We found that all cell lines and primary cells expressed TLR1, TLR3, TLR4, TLR7, TLR8, and TLR9 mRNA and protein. TLR2 and TLR5 were expressed by the majority of HMCLs at mRNA but were not detectable at protein level, while primary samples showed a low level of TLR2, TLR3 and TLR5 protein expression. Our results indicate that MM cells express a broad range of TLRs with a degree of disparity between gene and protein expression pattern. The clear expression of TLRs in MM cells indicates a propensity for responding to tumor-induced inflammatory signals, which seem inevitable in the MM bone marrow environment.     

R-Ras Protein Inhibits Autophosphorylation of Vascular Endothelial Growth Factor Receptor 2 in Endothelial Cells and Suppresses Receptor Activation in Tumor Vasculature

Abnormal angiogenesis is associated with a broad range of medical conditions, including cancer. The formation of neovasculature with functionally defective blood vessels significantly impacts tumor progression, metastasis, and the efficacy of anticancer therapies. Vascular endothelial growth factor (VEGF) potently induces vascular permeability and vessel growth in the tumor microenvironment, and its inhibition normalizes tumor vasculature. In contrast, the signaling of the small GTPase R-Ras inhibits excessive angiogenic growth and promotes the maturation of regenerating blood vessels. R-Ras signaling counteracts VEGF-induced vessel sprouting, permeability, and invasive activities of endothelial cells. In this study, we investigated the effect of R-Ras on VEGF receptor 2 (VEGFR2) activation by VEGF, the key mechanism for angiogenic stimulation. We show that tyrosine phosphorylation of VEGFR2 is significantly elevated in the tumor vasculature and dermal microvessels of VEGF-injected skin in R-Ras knockout mice. In cultured endothelial cells, R-Ras suppressed the internalization of VEGFR2, which is required for full activation of the receptor by VEGF. Consequently, R-Ras strongly suppressed autophosphorylation of the receptor at all five major tyrosine phosphorylation sites. Conversely, silencing of R-Ras resulted in increased VEGFR2 phosphorylation. This effect of R-Ras on VEGFR2 was, at least in part, dependent on vascular endothelial cadherin. These findings identify a novel function of R-Ras to control the response of endothelial cells to VEGF and suggest an underlying mechanism by which R-Ras regulates angiogenesis.     

CCR2介导MCP-1诱导的人内皮细胞凋亡

本室前期工作发现,单核细胞趋化蛋白-1(MCP-1)可诱导人内皮细胞(hVECs)凋亡.为进一步揭示MCP-1诱导凋亡分子机理,首先观察MCP-1对hVECs CC类趋化因子受体2(C-C motifchemokine receptor-2,CCR2)蛋白表达的影响.Western印迹结果显示,MCP-1以剂量依赖方式诱导CCR2在hVECs的表达.以脂质体为载体的CCR2反义寡核苷酸序列转染hVECs后,激光共聚焦显微镜及膜联蛋白(annexin)V-FITC/PI双染流式细胞术显示,CCR2反义寡核苷酸转染hVECs48h后可明显降低CCR2蛋白质的表达(P0.05),抑制MCP-1诱导的hVECs凋亡(P0.01).反义CCR2抑制凋亡结果与加入CCR2阻断剂RS102895后细胞凋亡测定结果一致.上述结果表明,MCP-1的主要受体CCR2介导了MCP-1诱导的hVECs凋亡.     

Antagonism of Bradykinin B2 Receptor Prevents Inflammatory Responses in Human Endothelial Cells by Quenching the NF-kB Pathway Activation

Background

Bradykinin (BK) induces angiogenesis by promoting vessel permeability, growth and remodeling. This study aimed to demonstrate that the B2R antagonist, fasitibant, inhibits the BK pro-angiogenic effects.

Methodology

We assesed the ability of fasibitant to antagonize the BK stimulation of cultured human cells (HUVEC) and circulating pro-angiogenic cells (PACs), in producing cell permeability (paracellular flux), migration and pseocapillary formation. The latter parameter was studied in vitro (matrigel assay) and in vivo in mice (matrigel plug) and in rat model of experimental osteoarthritis (OA). We also evaluated NF-κB activation in cultured cells by measuring its nuclear translocation and its downstream effectors such as the proangiogenic ciclooxygenase-2 (COX-2), prostaglandin E-2 and vascular endothelial growth factor (VEGF).

Principal findings

HUVEC, exposed to BK (1–10 µM), showed increased permeability, disassembly of adherens and tight-junction, increased cell migration, and pseudocapillaries formation. We observed a significant increase of vessel density in the matrigel assay in mice and in rats OA model. Importantly, B2R stimulation elicited, both in HUVEC and PACs, NF-κB activation, leading to COX-2 overexpression, enhanced prostaglandin E-2 production. and VEGF output. The BK/NF-κB axis, and the ensuing amplification of inflammatory/angiogenic responses were fully prevented by fasitibant as well as by IKK VII, an NF-κB. Inhibitor.

Conclusion

This work illustrates the role of the endothelium in the inflammation provoked by the BK/NF-κB axis. It also demonstates that B2R blockade by the antaogonist fasibitant, abolishes both the initial stimulus and its amplification, strongly attenuating the propagation of inflammation.     

Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC) Damages

Here, we studied the underlying mechanism of aldosterone (Aldo)-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L) inhibited human umbilical vein endothelial cells (HUVEC) survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18) production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P), an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS) inhibitor PDMP or the ceramide (C6) potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR) antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1) is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo.     

Mycobacterium paratuberculosis CobT Activates Dendritic Cells via Engagement of Toll-like Receptor 4 Resulting in Th1 Cell Expansion

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne disease in animals and MAP involvement in human Crohn disease has been recently emphasized. Evidence from M. tuberculosis studies suggests mycobacterial proteins activate dendritic cells (DCs) via Toll-like receptor (TLR) 4, eventually determining the fate of immune responses. Here, we investigated whether MAP CobT contributes to the development of T cell immunity through the activation of DCs. MAP CobT recognizes TLR4, and induces DC maturation and activation via the MyD88 and TRIF signaling cascades, which are followed by MAP kinases and NF-κB. We further found that MAP CobT-treated DCs activated naive T cells, effectively polarized CD4⁺ and CD8⁺ T cells to secrete IFN-γ and IL-2, but not IL-4 and IL-10, and induced T cell proliferation. These data indicate that MAP CobT contributes to T helper (Th) 1 polarization of the immune response. MAP CobT-treated DCs specifically induced the expansion of CD4⁺/CD8⁺CD44^highCD62L^low memory T cells in the mesenteric lymph node of MAP-infected mice in a TLR4-dependent manner. Our results indicate that MAP CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion in a TLR4-dependent cascade, suggesting that MAP CobT potentially links innate and adaptive immunity against MAP.