Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

Background

Pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs), recognize microbial components and trigger a host defense response. Respiratory tract infections are common causes of asthma exacerbations, suggesting a role for PRRs in this process. The present study aimed to examine the expression and function of PRRs on human airway smooth muscle cells (HASMCs).

Methods

Expression of TLR, NLR and RLR mRNA and proteins was determined using real-time RT-PCR, flow cytometry and immunocytochemistry. The functional responses to ligand stimulation were investigated in terms of cytokine and chemokine release, cell surface marker expression, proliferation and proteins regulating the contractile state.

Results

HASMCs expressed functional TLR2, TLR3, TLR4, TLR7 and NOD1. Stimulation with the corresponding agonists Pam₃CSK₄, poly(I:C), LPS, R-837 and iE-DAP, respectively, induced IL-6, IL-8 and GM-CSF release and up-regulation of ICAM-1 and HLA-DR, while poly(I:C) also affected the release of eotaxin and RANTES. The proliferative response was slightly increased by LPS. Stimulation, most prominently with poly(I:C), down-regulated myosin light chain kinase and cysteinyl leukotriene 1 receptor expression and up-regulated β₂-adrenoceptor expression. No effects were seen for agonist to TLR2/6, TLR5, TLR8, TLR9, NOD2 or RIG-I/MDA-5.

Conclusion

Activation of TLR2, TLR3, TLR4, TLR7 and NOD1 favors a synthetic phenotype, characterized by an increased ability to release inflammatory mediators, acquire immunomodulatory properties by recruiting and interacting with other cells, and reduce the contractile state. The PRRs might therefore be of therapeutic use in the management of asthma and infection-induced disease exacerbations.     

A Thermodynamic Funnel Drives Bacterial Lipopolysaccharide Transfer in the TLR4 Pathway

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Molecular and Cellular Response Profiles Induced by the TLR4 Agonist-Based Adjuvant Glucopyranosyl Lipid A

Background

Toll-like receptor (TLR)4 agonists are known potent immunostimulatory compounds. These compounds can be formulated as part of novel adjuvants to enhance vaccine medicated immune responses. However, the contribution of the formulation to the innate in vivo activity of TLR4 agonist compounds is not well understood.

Methodology and Principal Findings

We evaluated synthetic TLR4 agonist Glucopyranosyl Lipid A (GLA) for its effects on molecular and cellular innate immune responses in the murine model. Microarray techniques were used to compare the responses to GLA in an aqueous formulation or in an oil-in-water Stable Emulsion formulation (GLA-SE) versus either SE alone or the mineral salt aluminum hydroxide (alum) at the muscle injection site over multiple timepoints. In contrast to the minimal gene upregulation induced by SE and alum, both GLA and GLA-SE triggered MyD88- and TRIF-dependent gene expression. Genes for chemokines, cytokine receptors, signaling molecules, complement, and antigen presentation were also strongly upregulated by GLA and GLA-SE. These included chemokines for T_H1-type T cells (CXCL9 and CXCL10) and mononuclear leukocytes (CCL2, CCL3) among others. GLA-SE induced stronger and more sustained gene upregulation than GLA in the muscle; GLA-SE induced genes were also detected in local draining lymph nodes and at lower levels in peripheral blood. Both GLA and GLA-SE resulted in increased cellular trafficking to the draining lymph nodes and upregulated MHC molecules and ICAM1 on local dendritic cells. GLA and GLA-SE transiently upregulated circulating MCP-1, TNFα, IFNγ and IP-10 in blood.

Conclusions/Significance

While GLA and GLA-SE activate a large number of shared innate genes and proteins, GLA-SE induces a quantitatively and qualitatively stronger response than GLA, SE or alum. The genes and proteins upregulated could be used to facilitate selection of appropriate adjuvant doses in vaccine formulations.     

Comparison of Cytokine Expression in Human PBMCs Stimulated with Normal and Heat-Shocked Lactobacillus plantarum Cell Lysate

Probiotics and Antimicrobial Proteins - Regulation of immune responses is among the beneficial effects of probiotic bacteria on human health. In this study, we aim to investigate the effect of...     

人ING4的基因克隆及真核表达

目的克隆人生长抑制因子家族（inhibitor of growth famility member4,ING4）基因,构建其真核表达载体pEGFP—ING4。方法提取人胎盘总RNA,经RT—PCR扩增出ING4 cDNA,克隆至pEGFP—C2载体,构建的真核表达载体pEGFP—ING4用双酶切、基因测序进行序列鉴定;转染MCF-7细胞用荧光显微镜和免疫组化检测重组质粒的表达。结果RT—PCR产物为750bp的条带,双酶切和基因测序正确,转染可见目的蛋白融合表达。结论从人胎盘组织中成功克隆了ING4基因并构建其真核表达质粒在人MCF-7细胞中表达,为进一步研究1NG4基因的作用及抗肿瘤机制奠定了基础。     

衰老对2种猕猴血液基因表达的影响及与人类的对比

衰老是导致免疫功能和代谢活动下降的危险因素.作为最常用的非人灵长类动物模型,猕猴属Macaca被用来探索与衰老过程相关的因素.为了比较2种猕猴和人类在衰老过程基因表达变化的异同,本研究收集比较了3个物种的血液转录组.在藏酋猴M.thibetana(TM)、普通猕猴M.mulatta(CR)和人类中分别获得了2523个、...     

人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog 基因 表达的比较

目的:比较通过慢病毒方法获得的人诱导多能性干细胞(iPSCs)与人胚胎干细胞(hESCs)分化过程中全能性基因Oct4、Nanog的表达变化。方法:收集分化不同时间点的拟胚体(EBs),检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果:iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷(SCID)小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论:通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog基因表达的比较

目的：比较通过慢病毒方法获得的人诱导多能性干细胞（iPSCs）与人胚胎干细胞（hESCs）分化过程中全能性基因Oct4、Nanog的表达变化。方法：收集分化不同时间点的拟胚体（EBs）,检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果：iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷（SCID）小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论：通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

Structural Motifs Encoded by Individual Exons of the Human Neurokinin-1 Receptor Gene Interact Differentially with Selective Agonists and Antagonists

Abstract: Three chimeric receptors were constructed by exchanging exons between human neurokinin NK₁ and NK₃ receptor genes. The N-terminal sequences of these chimeric receptors are encoded by exon 1, exon 1–2, or exon 1–3 of the NK₁ receptor gene, whereas the remaining C-terminal sequences of these chimeric receptors are encoded by corresponding exons of the human NK₃ receptor gene. Substance P bound with high affinities to all three chimeric receptors, suggesting that in addition to the common structures composed of conserved amino acid residues among neurokinin receptors, structural elements encoded by the first exon of the human NK₁ receptor gene may also play an important role for substance P binding. On the contrary, potent NK₁ antagonists L703,606 and SR140,333 did not show any detectable binding to these chimeric receptors. In accordance, sequences encoded by exon 4, and possibly exon 5, are likely to contain important structural motifs that may directly or indirectly influence the binding of these antagonists. Further comparison of the binding affinities of highly selective NK₁ agonists, [Sar⁹,Met(O₂)¹¹]substance P, substance P methyl ester, and septide, revealed that each agonist may interact differently with the human NK₁ receptor. These results show that the exon-exchanging technique can be a useful tool for studying structure-function relationships of receptors in which exon-intron junctions are fully conserved among receptor subtypes.     

A Comparison of Collection Techniques for Gene Expression Analysis of Human Oral Taste Tissue

Variability in human taste perception is associated with both genetic and environmental factors. The influence of taste receptor expression on this variability is unknown, in part, due to the difficulty in obtaining human oral tissue that enables quantitative expression measures of taste genes. In a comparison of six current techniques (Oragene RNeasy Kit, Isohelix swab, Livibrush cytobrush, tongue saliva, cheek saliva collection, and fungiform papillae biopsy), we identify the fungiform papillae biopsy is the optimal sampling technique to analyse human taste gene expression. The fungiform papillae biopsy resulted in the highest RNA integrity, enabling amplification of all the assessed taste receptor genes (TAS1R1, TAS1R2, TAS1R3, SCNN1A and CD36) and taste tissue marker genes (NCAM1, GNAT3 and PLCβ2). Furthermore, quantitative expression was observed in a subset of taste genes assessed from the saliva collection techniques (cheek saliva, tongue saliva and Oragene RNA kit). These saliva collection techniques may be useful as a non-invasive alternative sampling technique to the fungiform papillae biopsy. Identification of the fungiform papillae biopsy as the optimal collection method will facilitate further research into understanding the effect of gene expression on variability in human taste perception.     

Lipopolysaccharide activates NF-kappaB by TLR4-Bcl10-dependent and independent pathways in colonic epithelial cells

In colonic epithelium, one of the pathways of lipopolysaccharide (LPS) activation of NF-kappaB and IL-8 is via Toll-like receptor (TLR)4, MyD88, IRAK1/4, and B-cell CLL/lymphoma 10 (Bcl10). However, this innate immune pathway accounts for only approximately 50% of the NF-kappaB activation, so additional mechanisms to explain the LPS-induced effects are required. In this report, we identify a second pathway of LPS-induced stimulation, mediated by reactive oxygen species (ROS), in human colonic epithelial tissue cells in tissue culture and in ex vivo mouse colonic tissue. Measurements of IL-8, KC, Bcl10, phospho-IkappaBalpha, nuclear NF-kappaB, and phosphorylated Hsp27 were performed by ELISA. The TLR4-Bcl10 pathway was inhibited by Bcl10 siRNA and in studies with colonic tissue from the TLR4-deficient mouse. The ROS pathway was inhibited by Tempol, a free radical scavenger, or by okadaic acid, an inhibitor of Hsp27 dephosphorylation by protein phosphatase 2A (PP2A). The ROS pathway was unaffected in the TLR4-deficient tissue or by silencing of Bcl10. The combination of exposure to the free radical scavenger Tempol and of TLR4 or Bcl10 suppression was required to completely inhibit the LPS-induced activation. The ROS pathway was associated with dephosphorylation of Hsp27. LPS appears to activate both the regulatory component of the IkappaBalpha-kinase (IKK) signalosome through Bcl10 interaction with Nemo (IKKgamma) and the catalytic component through Hsp27 interaction with IKKbeta. Since LPS exposure is associated with septic shock and the systemic inflammatory response syndrome, distinguishing between these two pathways of LPS activation may facilitate new approaches to prevention and treatment.     

人Toll样受体9基因启动子区的生物信息学分析

目的:探讨人Toll样受体9(TLR9)基因启动子区序列特征。方法:利用生物信息学技术预测人TLR9基因启动子区域、转录因子结合位点和CpG岛分布。结果:人TLR9基因启动子区有1434个转录因子结合位点,人和小鼠保守区域内存在23个共同的转录因子结合位点,人TLR9基因启动子区包含长572 bp的CpG岛。结论:人TLR9基因启动子区相关生物信息学的研究,提高了针对启动子的研究效率,并为预测基因启动子的功能提供了重要信息。     

人趋化因子受体CXCR4基因的克隆及原核表达

目的:构建人CXCR4原核表达载体并在大肠杆菌中进行表达。方法:从健康人外周血单个核细胞提取总RNA,以RT-PCR获得CXCR4基因全长1059bp的完整编码序列,将其克隆入载体pMD-18T中,经限制性内切酶和菌落PCR分析并测序证实的阳性重组子与表达载体pET-28a( )连接并转化大肠杆菌BL21(DE3)。结果:12%SDS-PAGE分析表明,在30℃以IPTG诱导获得分子量为43ku的His-CXCR4融合蛋白表达带,诱导4h后此蛋白表达量约为全菌总蛋白的25%。结论:成功获得人CXCR4基因融合蛋白。     

CREB4基因的表达谱分析和功能初步研究

cAMP反应元件结合蛋白(cAMPresponseelement-bindingproteins,CREB)是一个哺乳动物转录因子家族,通过cAMP反应元件(cAMPresponseelement,CRE)介导cAMP和钙离子依赖性基因表达。CREB4是CREB转录家族的新成员。人肿瘤MTCpanel结果显示,CREB4在人肺癌LX-1、结肠腺癌CX-1、前列腺癌PC-3、结肠癌G1-112和胰腺癌G1-103中有表达。构建表达CREB4-LexA和CREB215~395aa-LexA的pLexA融合质粒分别转化含p8opLacZ报告质粒的酵母EGY48菌株,诱导表达后发现CREB4蛋白为转录激活因子,N端决定其转录激活活性。亚细胞定位结果显示,全长CREB4蛋白定位于细胞质,而缺失C端假定转膜结构域的CREB41~275aa蛋白突变体则转移至细胞核内。表达谱结果显示CREB4蛋白可能在人多种肿瘤组织的基因表达调控中起作用,其C端假定的转膜结构域与其转录激活功能密切相关。