微小隐孢子虫IId亚型早期感染对HCT-8细胞TLRs的影响

目的 研究微小隐孢子虫IId亚型(中国流行株)感染对HCT-8细胞TLRs的影响。方法 微小隐孢子虫IId亚型感染HCT-8细胞4 h及12 h后,提取细胞RNA,利用qRT-PCR方法检测TLRs的表达水平,并通过生物信息学分析表达差异显著的miRNAs与TLRs的关系。结果 HCT-8细胞表达TLR1-TLR10 10种TLRs,并且微小隐孢子虫IId亚型早期感染(4 h和12 h)导致TLR2和TLR4表达量较对照组上调有统计学意义,其它8种TLRs较对照组表达变化无统计学意义。生物信息学分析表明TLR2和TLR4 并不是表达差异显著的miRNAs靶基因,但这些表达差异显著的miRNAs可以靶向27个TLRs信号通路基因。结论 TLR2和TLR4在微小隐孢子虫IId亚型感染HCT-8细胞中可能发挥着一定的作用,差异表达miRNAs的预测靶向中有TLR信号通路相关基因。     

通络止痛方对不同程度膝骨关节炎组织形态学和固有免疫的影响

[目的]评估通络止痛方对不同程度膝骨关节炎滑膜和软骨的组织形态学及固有免疫的影响。[方法]利用经典Hulth法建立轻度、中度、重度大鼠KOA动物模型,运用超声促透通络止痛方的方法对大鼠KOA模型进行干预,通过观察滑膜及软骨形态学变化确定病变程度,并对软骨进行Mankin’s评分。利用免疫荧光双标技术,观察软骨和滑膜上参与结构破坏的主要蛋白TLR4、NF-κB、MyD88表达量的变化。[结果]第2、4、8周组各组内通络止痛方干预组滑膜和软骨损伤程度较对照组明显减轻。在固有免疫中,各组内通络止痛方干预组的TLR4、NF-κB、MyD88的表达较对照组减弱。[结论]通络止痛方对不同程度膝骨关节炎均有抑制作用,证明通络止痛方可以通过抑制TLRs/MyD88信号通路相关原件的表达,抑制膝骨关节炎的发生和发展。     

The solute carrier SLC15A4 is required for optimal trafficking of nucleic acid–sensing TLRs and ligands to endolysosomes

A function-impairing mutation (feeble) or genomic deletion of SLC15A4 abolishes responses of nucleic acid–sensing endosomal toll-like receptors (TLRs) and significantly reduces disease in mouse models of lupus. Here, we demonstrate disease reduction in homozygous and even heterozygous Slc15a4 feeble mutant BXSB male mice with a Tlr7 gene duplication. In contrast to SLC15A4, a function-impairing mutation of SLC15A3 did not diminish type I interferon (IFN-I) production by TLR-activated plasmacytoid dendritic cells (pDCs), indicating divergence of function between these homologous SLC15 family members. Trafficking to endolysosomes and function of SLC15A4 were dependent on the Adaptor protein 3 (AP-3) complex. Importantly, SLC15A4 was required for trafficking and colocalization of nucleic acid–sensing TLRs and their ligands to endolysosomes and the formation of the LAMP2⁺VAMP3⁺ hybrid compartment in which IFN-I production is initiated. Collectively, these findings define mechanistic processes by which SLC15A4 controls endosomal TLR function and suggest that pharmacologic intervention to curtail the function of this transporter may be a means to treat lupus and other endosomal TLR-dependent diseases.

Engagement of endosomal toll-like receptors (TLRs)  by self-nucleic acids and, in certain instances, microbe-derived nucleic acids is central to the pathogenesis of lupus and likely many other autoimmune diseases (1–3). Consequently, efforts have sought to identify the relevant innate and adaptive cellular components and the mechanistic pathways involved in the trafficking of these inducer molecules into the appropriate subcellular compartments where endosomal TLR-mediated innate responses are initiated. Thus, it has been shown that type I interferon (IFN-I) and other proinflammatory cytokines constitute major disease mediators, plasmacytoid dendritic cells (pDCs) and B cells are required (4–7), and certain trafficking molecules, such as UNC-93B1, Adaptor protein 3 (AP-3), and BLOC-1/2, and the histidine–peptide cotransporter SLC15A4 are needed for optimal responses by endosomal TLRs (5, 8, 9). Among these molecules, SLC15A4 has received particular attention because it is expressed in both pDCs and B cells and may be susceptible to pharmacologic intervention.Our early studies with congenic lupus-predisposed C57BL/6 Fas^lpr mice carrying the function-impairing feeble mutation of Slc15a4 (5) and subsequent studies by others in the Slc15a4-deleted pristane model (10) and in the spontaneous NZB/W F1 lupus model (11) all showed significant disease reduction. These studies complemented earlier indirect evidence from genome-wide association studies that SLC15A4 may be one of the multitudes of loci contributing to systemic lupus erythemasosus (SLE) and other autoimmune syndromes (12–16).Despite these findings, the mechanistic processes by which SLC15A4 affects the function of nucleic acid–sensing TLRs remained unclear. An early study with mouse B cells presented evidence that the absence of SLC15A4 disrupts the metabolic mammalian target of rapamycin (mTOR) pathway, presumably by impairing vacuolar ATPase (v-ATPase) integrity, thereby leading to failure of the interferon regulatory factor (IRF) 7–IFN-I circuit and defective endolysosomal TLR signaling (10). In support of this finding, others have previously shown that mTOR complex 1 (mTORC1) senses lysosomal amino acids through a mechanism that requires v-ATPase (17). Furthermore, inference of mTOR involvement in Slc15a4 signaling was also made in studies with the human CAL-1 pDC line, and an effect on autophagy was suggested (18). Although these processes might occur within the confines of the endolysosomes, as we have previously hypothesized (5, 9), malfunction-inducing mutation or deletion of SLC15A4 may also compromise the trafficking of TLR and ligands into the late endosomes and/or affect the generation of subcellular organelles required for optimal recognition of nucleic acids. Here, we report the results of our investigation using high- and enhanced-resolution confocal microscopy and intracellular localization analysis techniques toward defining the potential effects of SLC15A4 in these processes as they may be manifested in primary pDCs.     

TLR4与肿瘤的研究进展

Toll样受体是近年来发现的Ⅰ型跨膜受体.在已发现的11种TLR中,TLR4是研究热点之一.活化的TLR4通过MyD88、丝裂原相关蛋白介导信号转导通路,诱导免疫反应的发生.TLR4在很多种肿瘤细胞中都有表达.TLR4通过不同的信号途径促进炎症和肿瘤的发生和发展,并在癌性疼痛和肿瘤免疫逃逸中起重要作用.TLR4可能成为一个潜在的肿瘤靶向治疗目标.     

蜕皮甾酮对口腔扁平苔藓上皮细胞TLRs/NF-KB信号通路的调节作用

目的：研究Toll样受体/核转录因子-kB （TLRs/NF-kB）信号通路在口腔扁平苔藓（oral lichen plus,OLP）病理过程中的作用,探讨说皮甾_ （ecdysterone, EDS）在OLP治疗中的作用效应。方法： （1）脂多糖（LPS）刺激人口腔黏膜角质形成细胞（HOK）建立体外OLP炎症模型,采用Real time RT- PCR检测NF-/κB p65,TLR4和肿瘤坏死因子-a（TNF-a）mRNA在该模型中的表达情况;（2）EDS干预 HOK细胞后,RT-PCR检测NF-K;Bp65、TLR4和TNF？amRNA的表达情况。结果：体外OLP炎症模 型中NF-κB p65、TLR4和TNF-amRNA表达量上调,且呈LPS浓度及作用时间依赖性,LPS浓度为 10吨/mL,作用6 h时表达量最高。EDS能够下调NF-κB p65、TLR4和TNF-amRNA的表达,EDS 最佳作用浓度是100 pg/mUPCO.Ol）。结论：EDS对OLP的抗炎及免疫调节作用与TLRs/NF-κB 信号通路密切相关,EDS可能通过抑制TLRs的激活,调控NF-/κB的表达,最终抑制OLP炎症介质活 化与黏膜损伤。     

小儿哮喘患儿经普米克令舒联合孟鲁司特治疗前后肺功能及免疫学细胞因子水平变化研究

目的 分析小儿哮喘患儿经普米克令舒联合孟鲁司特治疗前后肺功能及免疫学细胞因子水平变化.方法 选取2018年3月至2019年3月本院儿科收治的哮喘患儿86例,中途退出4例,最终纳入82例.按照治疗方法将其分为联合组和单一组,各组41例.单一组在常规治疗基础上增加孟鲁司特钠咀嚼片,联合组在单一组基础上增加普米克令舒.比较治...     

New DiaP277 analogue shifts DCs to tolerogenic,and modulates NF-Kβ1 to suppress autoreactive T lymphocytes in the type 1 diabetic mice

Abstract

Therapeutic efficacy of P277 against type 1 diabetes was extensively investigated and clinically evidenced. Clinical trials Phases I and II concluded promising results, while the data of P277 immunogenicity in Phase III trials represented weak responses that led to abolish medical use. But, a therapeutic performance of P277 cannot be forgotten. So, in order to exploit its therapeutic benefits and improve its immunogenicity, we developed a new analogue VP to optimize therapeutic efficacy and enhancing immunosuppressive modulations. However, new analogue was purified, and then used to immunize diabetic NOD mice to investigate antidiabetic effects through modulation of immunological status. So, DCs immune responses, relative TLRs, MyD88, and NF-Kβ1 mRNA expression on DCs and splenocytes under VP effect were tested. Circulating and intracellular cytokines were also evaluated at treated and non-treated mice. Splenic T lymphocytes proliferation (Th1 and Treg cells) were also determined. Results revealed that VP significantly down regulates DCs maturation through TLR2, TLR4, and MyD88 pathways. It also shifts DCs to a tolerogenic polarization through NF-Kβ1 pathway that mediates Th1 immunosuppression and enhances iTreg expanding in type1diabetes mice. Meanwhile, we noticed that VP significantly enhances iTreg CD25?+?FoxP3+ proliferation. In conclusion, VP showed promising immune potential to modulate immune regulatory responses and shifts DCs to suppress autoreactive Th1 cells which ameliorated immunosuppressive potency in the type1 diabetic mice.     

Deficiency in IRAK4 activity attenuates manifestations of murine Lupus

Interleukin‐1 receptor‐associated kinase (IRAK) 4 mediates host defense against infections. As an active kinase, IRAK4 elicits full spectra of myeloid differentiation primary response protein (MyD) 88‐dependent responses, while kinase‐inactive IRAK4 induces a subset of cytokines and negative regulators whose expression is not regulated by mRNA stability. IRAK4 kinase activity is critical for resistance against Streptococcus pneumoniae, but its involvement in autoimmunity is incompletely understood. In this study, we determined the role of IRAK4 kinase activity in murine lupus. Lupus development in BXSB mice expressing the Y chromosome autoimmunity accelerator (Yaa) increased basal and Toll‐like receptor (TLR) 4/7‐induced phosphorylation of mitogen‐activated protein kinases, p65 nuclear factor‐κB (NF‐κB), enhanced tumor necrosis factor (TNF)‐α and C‐C motif chemokine ligand (CCL) 5 gene expression in splenic macrophages, but decreased levels of Toll‐interacting protein and IRAK‐M, without affecting IRAK4 or IRAK1 expression. Mice harboring kinase‐inactive IRAK4 on the lupus‐prone Yaa background manifested blunted TLR signaling in macrophages and reduced glomerulonephritis, splenomegaly, serum anti‐nuclear antibodies, numbers of splenic macrophages, total and TNF‐α⁺ dendritic cells, activated T‐ and B‐lymphocytes, and lower TNF‐α expression in macrophages compared with lupus‐prone mice with functional IRAK4. Thus, IRAK4 kinase activity contributes to murine lupus and could represent a new therapeutic target.     

FSD-C10调节阿尔茨海默病双转基因小鼠炎性微环境

目的:探讨新型Rho激酶抑制剂FSD-C10对阿尔茨海默病(Alzheimer disease,AD)模型小鼠脑内炎性微环境的调节作用。方法:采用双转染人β-淀粉样蛋白前体(β-amyloid protein precursor,APP)695swe基因和人早老素1(presenilin-1,PS1)ΔE9突变基因的8月龄小鼠作为AD动物模型,随机分为模型组和FSD-C10治疗组,分别经腹腔注射生理盐水和FSD-C10(25 mg·kg~(-1)·d~(-1))持续治疗2个月,同月龄野生型小鼠作为正常对照组。应用Morris水迷宫(Morris water maze,MWM)实验检测小鼠学习和记忆能力。采用免疫组化和Western blot技术检测小鼠脑组织β-淀粉样蛋白(Aβ)、磷酸化Tau蛋白(p-Tau)、β位点APP剪切酶(BACE)、Toll样受体4(TLR-4)、磷酸化核因子κB(p-NF-κB)、诱导型一氧化氮合酶(i NOS)和精氨酸酶1(Arg-1)的表达。结果:与模型组相比,FSD-C10干预能显著改善APP/PS1双转基因小鼠学习和记忆能力,减少海马区Aβ1-42、p-Tau和BACE的表达,抑制脑内炎症信号通路TLRs/NF-κB轴TLR-4的表达和p-NF-κB的激活,减少i NOS的表达,增加Arg-1的表达。结论:FSD-C10干预能明显改善APP/PS1双转基因小鼠的学习和记忆能力,其机制可能是通过抑制TLRs/NF-κB信号通路激活,减少炎症因子的分泌及促进M1型炎性小胶质细胞向M2型抗炎小胶质细胞转化,从而改善APP/PS1双转基因小鼠脑组织炎症微环境。