miR-23a对LPS诱导小鼠巨噬细胞促炎性细胞因子表达的影响

 目的 探讨miR-23a对LPS诱导小鼠原代腹腔巨噬细胞促炎性细胞因子表达的影响。方法 Real-time PCR定量分析LPS刺激不同时间的腹腔巨噬细胞中miR-23a的表达以及瞬时转染miR-23a mimics和inhibitors的腹腔巨噬细胞中IL-1?、IL-6和TNF-?等促炎性细胞因子的表达;ELISA检测mRNA变化明显的细胞培养上清的IL-6的含量。结果 原代腹腔巨噬细胞在LPS刺激后,miR-23a的表达明显下调;在转染miR-23a mimics后,IL-6的表达和分泌明显升高 (P <0.05),但对IL-1?和TNF-?的表达没有明显影响。而转染miR-23a inhibitors后,则IL-6的表达水平受到抑制。结论 LPS刺激原代巨噬细胞可抑制miR-23a的表达;miR-23a可促进炎性细胞因子IL-6的表达,对IL-1?和TNF-?无显著影响。     

E3泛素连接酶RNF121对脂多糖诱导的巨噬细胞表达促炎性细胞因子的影响

目的探讨E3泛素连接酶RNF121对脂多糖(LPS)诱导的小鼠腹腔巨噬细胞表达促炎性细胞因子的调控作用。方法 LPS刺激小鼠腹腔巨噬细胞,Western blot检测RNF121的表达。用RNA干扰(si-RNF121)降低RNF121的表达,小鼠腹腔巨噬细胞经LPS刺激后,实时荧光定量PCR法检测TNF-α和IL-6的mRNA水平,ELISA检测细胞培养上清中TNF-α和IL-6的浓度,Western blot检测小鼠腹腔巨噬细胞中P65及磷酸化P65(p-P65)的表达水平,利用双荧光素酶报告基因法检测NF-κB的活性。结果 LPS诱导小鼠腹腔巨噬细胞后,RNF121的蛋白水平显著降低(P0.05),蛋白酶抑制剂MG132能够显著增加RNF121的蛋白水平。si-RNF121降低RNF121的表达后,给予LPS刺激小鼠腹腔巨噬细胞,IL-6和TNF-α的表达水平均显著下降(P0.05),转录因子P65的磷酸化(p-P65)水平及NF-κB的活性均显著降低(P0.05)。结论 E3泛素连接酶RNF121通过调控NF-κB的活性从而影响小鼠巨噬细胞促炎性细胞因子TNF-α及IL-6的表达。     

miR-146a对小鼠巨噬细胞IL-18表达的影响

目的:探讨miR-146a对小鼠单核-巨噬细胞株RAW264.7以及小鼠原代腹腔巨噬细胞Thl/Th2类细胞因子表达的影响。方法:体外培养的小鼠单核-巨噬细胞系RAW264.7细胞和腹腔新鲜分离的巨噬细胞分别瞬时转染miR-146a mimics、阴性对照mimics(NC mimics)、抑制性miR-146a(miR-146a inhibitor)和抑制性miR-146a阴性对照(NC in-hibitor)。转染后,利用real time PCR定量检测IL-18、IL-5和IL-10的表达情况。结果:RAW264.7细胞和原代腹腔巨噬细胞在转染miR-146a mimics后IL-18的表达水平明显降低(P<0.05),转染miR-146a inhibitor后IL-18的表达水平明显升高(P<0.05),但对IL-5和IL-10的表达,两种转染形式均没有影响。结论:巨噬细胞RAW264.7及原代巨噬细胞表达的miR-146a能够负向调控Thl类细胞因子IL-18的表达,但是不能调控Th2类细胞因子IL-5及IL-10的表达。     

雷公藤多苷对内毒素激活小鼠腹腔巨噬细胞分泌促炎症细胞因子的影响

目的：观察雷公藤多苷（TWP）对细菌内毒索（LPS）激活小鼠腹腔巨噬细胞（Mφ）分泌促炎症性细胞因子TNF-α和IL-6的影响，以探讨过度炎症反应中高细胞因子血症的调控药物和措施。方法：分离纯化小鼠腹腔Mφ，用LPS激活，与TWP共同孵育；以间接MTT法和ELISA法检测培养上清液中的TNF-α和IL-6的浓度。结果：TWP在浓度6．25～100μg/L，时间4～24小时范围内，对Mop产生TNF-α具有显著抑制作用（P〈0．01），呈剂量和时间依赖关系；TWP在浓度6．25～50μg／L范围内，时间12小时，对Mφ产生IL-6具有显著抑制作用（P〈0．01），呈剂量依赖关系。结论：TWP可以抑制LPS激活Mφ分泌促炎症性细胞因子的活性。     

Lnc-MALAT1减轻miR-217抑制 LPS诱导的大鼠肺泡巨噬细胞炎性反应

目的 探究长链非编码RNA转移相关肺腺癌转录本1(lnc-MALAT1)在脂多糖(LPS)诱导的大鼠肺泡巨噬细胞系(AMOs)炎性反应中的调控机制.方法 LPS处理AMOs建立细胞损伤模型,用脂质体法分别将pcDNA、pcDNA-MALAT1、miR-NC、miR-217 mimics、pcDNA-MALAT1与miR...     

MicroRNA-126基因敲减小鼠腹腔巨噬细胞功能的变化及意义

研究microRNA-126(miR-126)基因敲减小鼠腹腔巨噬细胞的功能变化,初步探讨其意义。采用real-time PCR检测LPS刺激前后野生型(wild type,WT)小鼠腹腔巨噬细胞miR-126表达变化,CCK8法检测其增殖情况;观察miR-126基因敲减(knock down,KD)小鼠腹腔巨噬细胞的形态变化,并用real-time PCR检测miR-126表达变化;CCK8法检测LPS刺激下miR-126KD小鼠腹腔巨噬细胞增殖变化;FACS检测巨噬细胞表面MHCⅡ类分子和CD86分子的表达变化;最后,real-time PCR检测巨噬细胞表达炎症因子IL-6、TGF-β和Ⅰ型精氨酸酶(arginase 1,Arg-1)等的变化情况。结果显示,WT小鼠腹腔巨噬细胞在LPS刺激后miR-126表达水平下调,显著低于未刺激组(P0.05),而增殖能力明显增强(P0.05);与WT小鼠相比,miR-126KD小鼠腹腔巨噬细胞的miR-126表达量明显下调(P0.05);形态上,WT小鼠腹腔巨噬细胞有较长伪足,多呈梭形,而miR-126KD小鼠腹腔巨噬细胞多呈圆形,细胞边缘光滑较少见伪足;LPS作用48h后,miR-126KD小鼠腹腔巨噬细胞的增殖能力明显强于WT小鼠(P0.05);且其膜MHCⅡ类分子和CD86分子表达也较WT小鼠显著上调(P0.05);LPS刺激下,miR-126KD小鼠腹腔巨噬细胞CCL-1表达显著增加,而IL-6、TGF-β和Arg-1的表达水平显著减少(P0.05)。这些结果提示,miR-126基因敲减可显著影响小鼠腹腔巨噬细胞的增殖能力和功能相关分子的表达,提示miR-126对小鼠腹腔巨噬细胞的功能具有重要的调控作用。     

乳腺癌中miR-146a对肿瘤相关巨噬细胞功能的影响

目的探讨miR-146a对肿瘤相关巨噬细胞(TAM)功能的影响及其在TAM中表达水平变化的调控机制。方法分别对比BALB/c小鼠4T1移植瘤TAM和腹腔巨噬细胞(PEC)、人乳腺癌组织TAM和外周血单核细胞,q-PCR法检测miR-146a的表达变化。转染miR-146a antagomir的巨噬细胞与4T1混合接种,确定TAM中miR-146a表达对肿瘤发生发展的影响。结果 miR-146a在荷瘤小鼠(P0.05)和人乳腺癌(P0.01)相关巨噬细胞中显著下调。miR-146a inhibitor明显抑制肿瘤的生长。结论 TAM中miR-146a可能通过其对巨噬细胞的调控进而促进肿瘤生长。     

人精浆免疫抑制作用对小鼠巨噬细胞的影响

SF_1是正常人精浆经Sephadex G100分离所得的第一组分。采用体内及体外途径,将SF_1作用于Balb/c小鼠巨噬细胞,实验证明SF_1对小鼠两种来源的巨噬细胞—腹腔巨噬细胞(PEC—Mφ)及骨髓细胞经体外培养衍生的巨噬细胞(BM—Mφ)均有明显的抑制作用,表现为Mφ吞噬抗体致敏羊红细胞(EA)的吞噬率及吞噬指数(PI)均明显低于对照组(P<0.01),並且抑制程度随SF_1浓度升高而逐渐明显;SF_1作用后的Mφ表面Fc受体及C_3b受体也明显受抑,表现为EA—花环及Yc—花环形成率降低(P<0.01)。此外,SF_1作用后的Mφ还表现表面皱褶减少及胞内空泡增加等超微结构的形态学改变。     

胞内寄生菌对巨噬细胞免疫逃逸的研究进展

病原微生物在感染宿主后,巨噬细胞作为主要的免疫哨兵细胞首先识别并吞噬入侵的病原体,而入侵的病原体可发展多种策略逃避巨噬细胞杀伤,并适应宿主细胞内环境,在胞内复制和存活。现对胞内感染常见细菌和真菌对巨噬细胞免疫逃逸的策略进行概述。     

重组卡介苗对小鼠巨噬细胞活性及细胞因子产生的影响

目的 将ｒＢＣＧ－ＩＬ－２应用于动脉实验,观察其对肿瘤生长和免疫功能的影响。方法 采用基因工程技术构建ｒＢＣＧ－ＩＬ－２,用黑色素瘤细胞株Ｂ１６接种于小鼠小腿外侧皮下,再用ｒＢＣＧ－ＩＬ－２局部治疗,２ｗｋ处死小鼠,用ＭＴＴＩ地检测腹腔巨噬细胞活必不学无术手淋巴细胞培养上清中ＩＬ－２,ＩＦＮ－ｒ和ＧＭ－ＣＳＦ的含量。结果 ｒＢＣＧ－ＩＬ－２对肿瘤生长的抑制作用与对照组比较有显著性差异Ｐ〈０．０１。     

RhoB induces the production of proinflammatory cytokines in TLR-triggered macrophages

Toll-like receptors (TLRs) are the primary sensors detecting conserved molecular patterns on microorganisms, thus acting as important components of innate immunity against invading pathogens. Many positive and negative regulators of TLR-triggered signaling have been identified. The Rho GTPase RhoB plays a key role in cell migration, division and polarity; however, the function and regulatory mechanisms of RhoB in TLR ligand-triggered innate immune responses remain to be investigated. Here, we report that the expression of RhoB is induced by TLR agonists (lipopolysaccharide (LPS), CpG, poly(I:C)) in macrophages. Knockdown of RhoB expression markedly decreased TLR ligand-induced activation of mitogen activated protein kinases and nuclear factor-κB (NF-κB), and the production of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1β in macrophages stimulated with TLR ligands. Furthermore, we demonstrated that RhoB interacts with major histocompatibility complex class II (MHCII) α chain, but not β chain, in endosomes of macrophages. Knockdown of MHCII expression greatly reduced the interaction of RhoB with Btk, and attenuated the induction of NF-κB and interferon β activity by RhoB upon LPS stimulation. These findings suggest that RhoB is a positive physiological regulator of TLRs signaling via binding to MHCII in macrophages, and therefore RhoB may be a potential therapeutic target in inflammatory diseases.     

Mycobacterium tuberculosis,macrophages, and the innate immune response: does common variation matter?

Summary:  Despite the discovery of the tuberculosis (TB) bacillus over 100 years ago and the availability of effective drugs for over 50 years, there remain a number of formidable challenges for controlling Mycobacterium tuberculosis (MTb). Understanding the genetic and immunologic factors that influence human susceptibility could lead to novel insights for vaccine development as well as diagnostic advances to target treatment to those who are at risk for developing active disease. Although a series of studies over the past 50 years suggests that host genetics influences resistance to TB, a comprehensive understanding of which genes and variants are associated with susceptibility is only partially understood. In this article, we review recent advances in our understanding of human variation of the immune system and its effects on macrophage function and influence on MTb susceptibility. We emphasize recent discoveries in human genetic studies and correlate these findings with efforts to understand how these variants alter the molecular and cellular functions that regulate the macrophage response to MTb.     

Interleukin‐32 production associated with biliary innate immunity and proinflammatory cytokines contributes to the pathogenesis of cholangitis in biliary atresia

Biliary atresia (BA) is thought to be associated with infections by viruses such as Reoviridae and is characterized histologically by fibrosclerosing cholangitis with proinflammatory cytokine‐mediated inflammation. Interleukin (IL)‐32 affects the continuous inflammation by increasing the production of proinflammatory cytokines. In this study, the role of IL‐32 in the cholangitis of BA was examined. Immunohistochemistry for IL‐32 and caspase 1 was performed using 21 samples of extrahepatic bile ducts resected from BA patients. Moreover, using cultured human biliary epithelial cells (BECs), the expression of IL‐32 and its induction on stimulation with a Toll‐like receptor [(TLR)‐3 ligand (poly(I:C)] and proinflammatory cytokines was examined. BECs composing extrahepatic bile ducts showing cholangitis expressed IL‐32 in BA, but not in controls. Caspase 1 was expressed constantly on BECs of both BA and control subjects. Furthermore, poly(I:C) and proinflammatory cytokines [(IL‐1β, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α] induced IL‐32 expression strongly in cultured BECs, accompanying the constant expression of TLR‐3 and caspase 1. Our results imply that the expression of IL‐32 in BECs was found in the damaged bile ducts of BA and induced by biliary innate immunity via TLR‐3 and proinflammatory cytokines. These findings suggest that IL‐32 is involved initially in the pathogenic mechanisms of cholangitis in BA and also plays an important role in the amplification and continuance of periductal inflammatory reactions. It is therefore tempting to speculate that inhibitors of IL‐32 could be useful for attenuating cholangitis in BA.     

Neutrophil secretion products regulate anti-bacterial activity in monocytes and macrophages

Macrophages represent a multi-functional cell type in innate immunity that contributes to bacterial clearance by recognition, phagocytosis and killing. In acute inflammation, infiltrating neutrophils release a wide array of preformed granule proteins which interfere functionally with their environment. Here, we present a novel role for neutrophil-derived granule proteins in the anti-microbial activity of macrophages. Neutrophil secretion obtained by antibody cross-linking of the integrin subunit CD18 (X-link secretion) or by treatment with N-Formyl-Met-Leu-Phe (fMLP secretion) induced a several-fold increase in bacterial phagocytosis by monocytes and macrophages. This response was associated with a rapid activation of the monocytes and macrophages as depicted by an increase in cytosolic free Ca(2+). Interestingly, fMLP secretion had a more pronounced effect on monocytes than the X-link secretion, while the opposite was observed for macrophages. In addition, polymorphonuclear cells (PMN) secretion caused a strong enhancement of intracellular reactive oxygen species (ROS) formation compared to incubation with bacteria. Thus, secretion of neutrophil granule proteins activates macrophages to increase the phagocytosis of bacteria and to enhance intracellular ROS formation, indicating pronounced intracellular bacterial killing. Both mechanisms attribute novel microbicidal properties to PMN granule proteins, suggesting their potential use in anti-microbial therapy.     

Enhanced natural killer activity and production of pro-inflammatory cytokines in mice selected for high acute inflammatory response (AIRmax)

Strains of mice with maximal and minimal acute inflammatory responsiveness (AIRmax and AIRmin, respectively) were developed through selective breeding based on their high- or low-acute inflammatory responsiveness. Previous reports have shown that AIRmax mice are more resistant to the development of a variety of tumours than AIRmin mice, including spontaneous metastasis of murine melanoma. Natural killer activity is involved in immunosurveillance against tumour development, so we analysed the number and activity of natural killer cells (CD49b(+)), T-lymphocyte subsets and in vitro cytokine production by spleen cells of normal AIRmax and AIRmin mice. Analysis of lymphocyte subsets by flow cytometry showed that AIRmax mice had a higher relative number of CD49b(+) cells than AIRmin mice, as well as cytolytic activity against Yac.1 target cells. The number of CD3(+) CD8(+) cells was also higher in AIRmax mice. These findings were associated with the ability of spleen cells from AIRmax mice in vitro to produce higher levels of the pro-inflammatory cytokines tumour necrosis factor-alpha, interleukin-12p40 and interferon-gamma but not the anti-inflammatory interleukin-10. Taken together, our data suggest that the selective breeding to achieve the AIRmax and AIRmin strains was able to polarize the genes associated with cytotoxic activity, which can be responsible for the antitumour resistance observed in AIRmax mice.     

Antiviral effect of ovotransferrin in mouse peritoneal macrophages by up-regulating type I interferon expression

Ovotransferrin (OVT) is one of the major proteins in avian albumen that exerts diverse biological activities. This study is aimed to investigate the antiviral effect and mechanism of OVT from the avian egg. Effects of OVT on vesicular stomatitis virus (VSV)-infected mouse peritoneal macrophage were detected by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and western blot. In OVT-pretreated mouse peritoneal macrophages, results showed that replication of VSV was significantly suppressed and production of type I interferon (IFN) was increased. OVT down-regulated ring finger protein 125 (RNF125) production and up-regulated retinoic-acid-inducible gene I (RIG-I) expression by the nuclear factor-κB signalling pathway, resulting in the enhancement of the production of type I IFN. It demonstrated that the antiviral effect of OVT on innate immunity is enhanced by promoting type I IFN expression, indicating its potential as a functional food ingredient.     

High serum miR-183 level is associated with the bioactivity of macrophage derived from tuberculosis patients

As a major health threat, tuberculosis (TB) is resistant against the current therapeutic strategies. Increasing evidence indicates that miRNAs are implicated in various disorders by affecting specific target genes. Recently, the association of miRNAs with TB has also been established by several studies, and their potentials in the prognosis and treatment of TB have also been verified. miR-183 is shown to promote the activation of macrophage through NF-κB pathway. However, it is still unclear if serum miR-183 can be used to assess the activity of TB-associated macrophage. This study was aimed to address this issue. We employed qPCR assay to detect the expression level of miR-183 in blood from TB patients and healthy individuals. miR-183 abundance was found to be increased in serum samples from TB patients, compared with healthy controls. Further analysis revealed that miR-183 level is positively associated with the activity of macrophages from TB patients, evidenced by their increased phagocytosis rates and enzyme activity in high serum miR-183 group. In conclusions, high level of serum miR-183 is associated with the activity of macrophage originating from TB patients.