鸟结核分枝杆菌感染巨噬细胞的分子免疫机制研究

目的:探讨鸟结核分枝杆菌感染巨噬细胞后产生的免疫防御分子机制.方法:通过抗酸染色分析巨噬细胞受M.avium感染后吞噬M.aviu的能力;并运用real-time PCR及流式细胞术从基因和蛋白水平检测巨噬细胞受M.avium感染后细胞CD80、CD86的表达情况;同时以ELISA方法检测M.avium感染巨噬细胞后细胞上清中IFN-γ、TNF-α含量.结果:巨噬细胞随着时间变化吞噬M.avium的量不断增加;real-time PCR与流式细胞术结果表明巨噬细胞在受M.avium感染后CD80、CD86基因与蛋白表达上调.同时ELISA检测结果表明IFN-γ、TNF-α在M.avium感染巨噬细胞后培养上清中含量增加.结论:巨噬细胞吞噬M.avium的能力随时间变化而增强.M.avium可促进巨噬细胞表面信号分子CD80、CD86在基因及蛋白水平表达表达增强,并促进巨噬细胞TNF-α、IFN-γ的分泌从而增强巨噬细胞的炎症反应.     

外泌体与结核分枝杆菌相互作用的研究进展

结核病是目前世界各国重点防控传染性疾病之一。外泌体(exosomes)是多种细胞分泌的一种含有特异性与非特异性蛋白成分的囊泡结构,是机体免疫细胞之间重要的信息传递介质,其在机体免疫反应中发挥着重要作用。外泌体能呈递表面抗原,激活多种细胞免疫因子,介导巨噬细胞杀伤结核杆菌的细胞免疫反应。外泌体与结核分枝杆菌相互作用研究已经成为当前重大研究热点,有望开发成预防结核病的新型疫苗。     

外泌体对M2型巨噬细胞的调控及其在心血管疾病中的作用①

外泌体是一种脂质微囊泡,可携带多种脂质、蛋白质、核酸,参与细胞间通讯,是心血管疾病的新型标志物和潜在治疗靶点。M2型巨噬细胞可抑制血管炎症反应、促进组织修复,在心血管疾病中发挥保护作用,其功能受多种因素调控。外泌体是近年逐渐受到关注的细胞对话新方式。本文根据外泌体来源进行分类,总结外泌体内不同物质对M2型巨噬细胞的调节作用、相关信号通路及其与心血管疾病的潜在关系。     

低氧对巨噬细胞外泌体的表达及其对骨肉瘤细胞的化疗抗性影响的体外研究

目的在体外探究低氧对巨噬细胞外泌体分泌的影响及对骨肉瘤细胞顺铂耐药性的改变。方法Transwell侵袭实验检测不同氧浓度(1%O2的低氧与常氧)条件下骨肉瘤细胞MG63对巨噬细胞的趋化能力;在低氧与常氧条件下将MG63与巨噬细胞进行共培养,流式细胞术及RT-PCR检测巨噬细胞的分化;分离纯化来源于不同氧浓度条件下培养的M2型巨噬细胞外泌体,利用透射电镜、纳米粒径、Western blot进行鉴定,双免疫荧光进行示踪MG63对外泌体的摄取;克隆形成实验及流式细胞术检测来源于不同氧浓度下的巨噬细胞外泌体对MG63的增殖、凋亡影响;CCK-8检测来源于不同氧浓度下的巨噬细胞外泌体对MG63顺铂耐药性的影响。结果低氧能够促进骨肉瘤细胞对巨噬细胞的趋化能力,并进一步诱导巨噬细胞向M2型分化;成功分离不同氧浓度下培养的M2巨噬细胞外泌体,且低氧能够显著上调巨噬细胞对外泌体的分泌;低氧能够明显上调巨噬细胞外泌体对骨肉瘤细胞增殖能力的促进作用,抑制骨肉瘤细胞发生凋亡,并上调骨肉细胞对顺铂的耐药性。结论在体外低氧能够促进骨肉瘤细胞对巨噬细胞的趋化能力,并进一步通过诱导巨噬细胞向M2型分化及提高M2型巨噬细胞外泌体的分泌,进而上调骨肉瘤细胞的增殖能力,抑制其凋亡,最终导致骨肉瘤细胞产生顺铂耐药性。     

肿瘤源性外泌体对巨噬细胞极化的调控作用的研究进展

单核巨噬细胞是一种功能多样、对不同的微环境信号应答表现出不同反应的集群。根据受到的不同刺激, 巨噬细胞可极化为经典活化型(M1)及替代活化型(M2), 是巨噬细胞功能表现的两个极端。核因子-κB、环氧合酶2、乏氧状态、原癌基因MYC、Toll样受体信号通路、Notch信号通路及细胞因子等密切参与肿瘤相关巨噬细胞发生M1-M2型别的转化。其中浸润在肿瘤组织中的巨噬细胞, 会受肿瘤产生的细胞因子的影响而表现出M2表型, 这些极化的巨噬细胞在促进肿瘤的发生与进展和破坏适应性免疫应答方面作用显著。肿瘤细胞来源的外泌体具有肿瘤细胞本身的特性, 可参与多种肿瘤发生、发展的多个过程。本文综述了来源于多种癌细胞的外泌体, 详细讨论了在肿瘤免疫微环境中, 肿瘤源性外泌体的内容物在调节巨噬细胞极化行为中的作用及其涉及的信号机制。     

M2型巨噬细胞外泌体诱导骨髓间充质干细胞的成骨分化北大核心

背景:目前研究表明,M2型巨噬细胞能够促进成骨分化并呈网络状调控,外泌体可携带大量信息参与细胞间的信号传导,M2型巨噬细胞来源外泌体是否能促进骨髓间充质干细胞的成骨分化,有待研究。目的:探究M2型巨噬细胞来源外泌体对骨髓间充质干细胞成骨分化的影响。方法:培养鼠源性巨噬细胞系RAW264.7与鼠骨髓间充质细胞系CP-M131,培养至第3代,应用白细胞介素4诱导巨噬细胞极化为M2型巨噬细胞,从M2型巨噬细胞培养上清中提取外泌体,然后用终质量浓度为0,30,60,90 mg/L的外泌体与骨髓间充质干细胞共培养72 h后收集样本,以及60 mg/L M2型巨噬细胞来源外泌体与骨髓间充质干细胞共培养24,48,72 h后收集样本,Western blot检测骨髓间充质干细胞中成骨相关因子RUNX2和碱性磷酸酶蛋白表达,茜素红染色检测矿物质沉积情况。结果与结论:①成骨相关因子RUNX2及碱性磷酸酶的表达水平与巨噬细胞外泌体质量浓度密切相关,与空白对照组比较,60 mg/L外泌体组RUNX2、碱性磷酸酶表达明显升高(P<0.05),干预72 h RUNX2、碱性磷酸酶表达明显升高(P<0.05);②茜素红实验结果表明,与空白对照组比较,60 mg/L外泌体组钙结节含量较高(P<0.05),干预72 h钙结节含量较高(P<0.05);③结果表明,M2型巨噬细胞分泌的外泌体能够诱导骨髓间充质干细胞向成骨细胞分化。     

M2型巨噬细胞来源外泌体对乳腺癌细胞系4T1干性特征的影响

目的探讨M2型巨噬细胞来源的外泌体对乳腺癌细胞系4T1细胞肿瘤干性特征的影响。方法用超速离心法分离M2型巨噬细胞来源的外泌体;用透射电子显微镜、纳米粒子追踪技术,Western blot等对其进行鉴定;然后以乳腺癌细胞系4T1为肿瘤模型,使用Dil染料标记M2型巨噬细胞来源的外泌体,通过免疫荧光技术观察其能否进入4T1细胞。此外,用流式细胞计量术、共培养体系和Transwell实验探究在M2型巨噬细胞来源的外泌体影响下的4T1细胞的干性特征。结果 M2型巨噬细胞在体外被成功诱导,分离并鉴定了M2型巨噬细胞来源的外泌体。通过免疫荧光,看到Dil标记的外泌体可成功进入4T1细胞。M2型巨噬细胞来源的外泌体与4T1细胞共孵育后,可明显增强4T1细胞的迁移能力(P0.001)和成球能力(P0.05);外泌体抑制剂GW4869可明显抑制4T1细胞的成球能力(P0.05)。同时,M2型巨噬细胞来源的外泌体可明显增加4T1细胞中CD44~(high)CD24~(low)细胞的比例(P0.001)。结论 M2型巨噬细胞来源的外泌体能够促进4T1肿瘤细胞的迁移和成球能力,并明显增加4T1细胞中肿瘤干细胞的比例。     

尿毒症毒素通过刺激巨噬细胞外泌体分泌miR-22促进心肌细胞自噬

目的:探讨在尿毒症毒素微环境下巨噬细胞外泌体分泌微小RNA-22(miR-22)对心肌细胞自噬的影响。方法:收集硫酸吲哚酚(IS)刺激后的巨噬细胞源性外泌体,并将其与H9c2细胞共培养,RT-qPCR检测巨噬细胞及其分泌的外泌体中miR-22的表达水平,CCK-8法检测H9c2细胞的活力,Western blot检测外泌体表面标志蛋白CD63及自噬相关蛋白LC3和P62的表达。结果:在IS刺激下,巨噬细胞中外泌体表面标志蛋白CD63的水平显著高于对照组(P<0. 05),同时IS组巨噬细胞及其分泌的外泌体中miR-22的水平较对照组显著上调(P<0. 01)。随着共培养体系中巨噬细胞外泌体浓度的增大,H9c2细胞的活力逐渐降低(P<0. 05),并且巨噬细胞外泌体的刺激降低了H9c2细胞P62的表达,并促进了LC3-Ⅰ向LC3-Ⅱ转化,这种变化呈剂量依赖性(P<0. 05)。在巨噬细胞外泌体刺激下,miR-22模拟物转染的H9c2细胞与相应阴性对照miRNA转染的细胞相比,具有更高的LC3-Ⅱ/LC3-Ⅰ比率(P<0. 05)及更低的P62蛋白表达量(P&...     

外泌体在新型冠状病毒感染和传播中作用的研究进展

外泌体在COVID-19的病理过程中起着重要作用，与其携带的核酸、蛋白质等物质，共同调控SARS-CoV-2的感染和传播。病毒感染细胞通过内体途径产生含SARS-CoV-2病毒颗粒的外泌体，介导病毒向健康细胞感染和传播，造成组织损伤和多器官功能障碍。同时，外泌体作为细胞间通讯的关键介质，起到免疫激活作用，促进机体产生免疫反应，抵抗病毒入侵。此外，外泌体协助病毒逃避机体免疫，是SARS-CoV-2再感染的潜在手段。总之，外泌体在新型冠状病毒感染和传播中的作用为其在诊断、防治COVID-19等方面的应用提供新思路。     

肝癌细胞外泌体诱导巨噬细胞M2极化后促进肝癌细胞迁移及侵袭

目的：探讨外泌体在肝细胞癌（HCC）细胞与巨噬细胞间是否存在活性物质交通作用，分析肿瘤微环境中肿瘤相关巨噬细胞（TAMs）与HCC细胞转移的关系及机制。方法：采用超速离心法从Huh-7和RAW264.7细胞的培养基中提取外泌体，Western blot和透射电镜进行外泌体鉴定；激光共聚焦显微镜对外泌体与受体细胞结合内化进行追踪；qRT-PCR及ELISA检测巨噬细胞表型变化；miRNA模拟物进行功能性实验验证其作用；细胞划痕及Transwell试验检测TAMs对HCC细胞迁移及侵袭能力的影响。结果：HCC细胞外泌体可被巨噬细胞内吞；肝癌细胞外泌体及miR-151模拟物处理的巨噬细胞M2型标志物CD206、Arg-1 mRNA表达及IL-10、TGF-β蛋白表达显著高于对照组（P<0.05）;TAMs外泌体处理组HCC细胞迁移及侵袭能力高于其他组（P<0.05）。结论：HCC细胞来源外泌体miR-151可诱导巨噬细胞极化为TAMs,TAMs对肝癌细胞迁移及侵袭有促进作用。     

Regulation of expression of major histocompatibility antigens by bovine macrophages infected with Mycobacterium avium subsp. paratuberculosis or Mycobacterium avium subsp. avium

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically very similar organisms; however, they differ markedly in their virulence for cattle. We evaluated the capacity of bovine macrophages infected with M. avium subsp. paratuberculosis or M. avium subsp. avium to express major histocompatibility complex (MHC) class I and class II antigens on their surface and to interact with primed autologous lymphocytes. Our results indicate that infection of bovine macrophages with M. avium subsp. paratuberculosis promoted the downregulation of MHC class I and class II molecules on the macrophage surface within 24 and 12 h, respectively. Alternatively, MHC class II expression by M. avium subsp. avium-infected macrophages was not detected until 24 h after infection, and the magnitude of the decrease was smaller. Decreased MHC class I expression by M. avium subsp. avium-infected macrophages was not detected. Unlike M. avium subsp. paratuberculosis-infected macrophages, M. avium subsp. avium-infected macrophages upregulated MHC class I and class II expression after activation by gamma interferon or tumor necrosis factor alpha. Further, M. avium subsp. avium-infected macrophages were lysed by primed autologous lymphocytes, whereas M. avium subsp. paratuberculosis-infected macrophages were not. Overall, the results support the hypothesis that the difference in the virulence of M. avium subsp. paratuberculosis and M. avium subsp. avium for cattle is dependent on a difference in the capacity of the organisms to suppress mycobacterial antigen presentation to T lymphocytes.     

Response to stimulation with recombinant cytokines and synthesis of cytokines by murine intestinal macrophages infected with the Mycobacterium avium complex.

Current evidence suggests that the gut is the chief portal of entry for organisms of the Mycobacterium avium complex (MAC) in AIDS patients. Bacterial invasion of intestinal mucosa presumably occurs through epithelial cells, and M cells in the Peyer's patches, where the bacteria have contact with immunocompetent cells such as macrophages and T and B lymphocytes. As mucosal macrophages are probably the first line of defense against MAC, we examined their ability to inhibit intracellular growth of MAC when properly stimulated. Mouse intestinal macrophages were purified, infected with MAC 101, serovar 1, and MAC 86-2686, serovar 16, and subsequently stimulated with recombinant tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage colony-stimulating factor (M-CSF). Viable intracellular bacteria were quantitated at 24 h after infection and again after 4 days of infection. Stimulation with TNF-alpha, IFN-gamma, and GM-CSF, but not M-CSF, was associated with mycobacteriostatic and/or mycobactericidal activity in macrophages. Treatment with 10(3) U of TNF-alpha, GM-CSF, and IFN-gamma per ml at 24 h prior to infection with MAC resulted in a significant enhancement in killing of MAC at 4 days after infection, compared with that observed for macrophages exposed to cytokines after infection. When stimulated with lipopolysaccharide or live MAC, intestinal macrophages had produced significantly less TNF-alpha and transforming growth factor beta than had splenic and peritoneal macrophages, although the levels of production of interleukin 6 and interleukin 10 among the three populations of cells were similar. Intestinal macrophages can be stimulated with cytokines to inhibit the intracellular growth of MAC, but they have differentiated abilities to produce cytokines which can modulate the anti-MAC immune response.     

Activation of macrophages and interference with CD4+ T-cell stimulation by Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subspecies avium

Mycobacterium avium subspecies paratuberculosis (M. ptb) and M. avium subspecies avium (M. avium) are closely related but exhibit significant differences in their interaction with the host immune system. The macrophage line, J774, was infected with M. ptb and M. avium and analysed for cytokine production and stimulatory capacity towards antigen-specific CD4+ T cells. Under all conditions J774 cells were activated to produce proinflammatory cytokines. No influence on the expression of major histocompatibility complex (MHC) class II, intracellular adhesion molecule-1 (ICAM-1), B7.1, B7.2 or CD40 was found. However, the antigen-specific stimulatory capacity of J774 cells for a CD4+ T-cell line was significantly inhibited after infection with M. ptb, but not with M. avium. When a T-cell hybridoma expressing a T-cell receptor identical to that of the T-cell line was used, this inhibition was not observed, suggesting that costimulation which is essential for the CD4+ T-cell line is influenced by the pathogenic bacterium M. ptb.     

Infection with Mycobacterium avium differentially regulates the expression of iron transport protein mRNA in murine peritoneal macrophages

Iron is an important element for the growth of microorganisms as well as in the defense of the host by serving as a catalyst for the generation of free radicals via the Fenton/Haber-Weiss reactions. The iron transporter natural resistance-associated macrophage protein 1 (Nramp1) confers resistance to the growth of a variety of intracellular pathogens including Mycobacterium avium. Recently several other proteins that are involved in iron transport, including the highly homologous iron transporter Nramp2 and the transferrin receptor-associated protein HFE (hereditary hemochromatosis protein), have been described. The relationship of these proteins to host defense and to the growth of intracellular pathogens is not known. Here, we report that infection with M. avium differentially regulates mRNA expression of the proteins associated with iron transport in murine peritoneal macrophages. Both Nramp1 and Nramp2 mRNA levels increase following infection, while the expression of transferrin receptor mRNA decreases. The level of expression of HFE mRNA remains unchanged. The difference in the expression of the mRNA of these proteins following infection or cytokine stimulation suggests that they may play an important role in host defense by maintaining a delicate balance between iron availability for host defense and at the same time limiting iron availability for microbial growth.     

Surfactant protein A enhances Mycobacterium avium ingestion but not killing by rat macrophages

Mycobacterium avium complex (MAC) is a significant cause of opportunistic infection in patients with acquired immunodeficiency syndrome. Although the major route of entry of MAC is via the gastrointestinal tract, MAC can infect humans through the respiratory tract and eventually encounter alveolar macrophages within the lung. Once in the lung, MAC can potentially interact with surfactant protein A (SP-A), an important component of the pulmonary innate-immune response. Previous work on other pulmonary pathogens including Mycobacterium bovis Bacillus Calmette-Guerin (BCG) suggests that SP-A participates in promoting efficient clearance of these organisms by alveolar macrophages. In the present study, we investigated the role of SP-A in clearance of MAC by cultured rat macrophages. SP-A bound to MAC organisms and enhanced the ingestion of the mycobacteria by macrophages. Infection of macrophages with SP-A-MAC complexes induced the production of nitric oxide (NO) and tumor necrosis factor-alpha. However, intracellular survival of MAC was not altered by preopsonization with SP-A. In addition, inhibitors of inducible NO synthase did not alter MAC clearance. These results suggest that SP-A can bind to and enhance the uptake of MAC by alveolar macrophages, similar to previous findings with BCG and Mycobacterium tuberculosis.However, unlike BCG and other pulmonary pathogens that are cleared effectively in the presence of SP-A via a NO-dependent pathway, macrophage-mediated clearance of MAC is not enhanced by SP-A.     

A pre-existing infection by Mycobacterium avium subsp. avium modulates anti-Cryptococcus neoformans and anti-Candida albicans activities in human macrophages

Mycobacterium avium is a facultative intracellular microorganism, able to survive and multiply within mammalian macrophages by circumventing antimicrobial mechanisms. In this study we hypothesize that pre-existing M. avium infection could result in macrophage superinfections by other microorganisms. We found that 24 h after ingestion of M. avium at a low multiplicity of infection, macrophages are unable to efficiently produce superoxide anions when over-stimulated with phorbol esters, and that the generation of oxidative burst is only partially restored 72 h after bacteria ingestion. We also demonstrate that intracellular killing of Cryptococcus neoformans is markedly impaired in human macrophages that have previously ingested M. avium (but not other bacteria such as Escherichia coli). This inhibitory effect is observed with live mycobacteria, but not when heat-inactivated bacteria are ingested. In contrast, when Candida albicans is given to macrophages instead of C. neoformans, an enhancement of intracellular killing is observed, suggesting that cytocidal mechanisms other than respiratory burst are involved in the anti- Candidacidal activity of macrophages.     

Differential responses of bovine macrophages to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically similar organisms; however, they differ in their virulence for cattle. M. avium subsp. paratuberculosis causes a chronic intestinal infection leading to a chronic wasting disease termed paratuberculosis or Johne's disease, whereas M. avium subsp. avium causes only a transient infection. We compared the response of bovine monocyte-derived macrophages to ingestion of M. avium subsp. paratuberculosis and M. avium subsp. avium organisms by determining organism survival, superoxide and nitric oxide production, and expression of the cytokines tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-8 (IL-8), IL-10, IL-12, and granulocyte-monocyte colony-stimulating factor (GM-CSF). Unlike M. avium subsp. paratuberculosis, macrophages were able to kill approximately half of the M. avium subsp. avium organisms after 96 h of incubation. This difference in killing efficiency was not related to differences in nitric oxide or superoxide production. Compared to macrophages activated with IFN-gamma and lipopolysaccharide, macrophages incubated with M. avium subsp. paratuberculosis showed greater expression of IL-10 and GM-CSF (all time points) and IL-8 (72 h) and less expression of IL-12 (72 h), IFN-gamma (6 h), and TNF-alpha (6 h). When cytokine expression by macrophages incubated with M. avium subsp. paratuberculosis was compared to those of macrophages incubated with M. avium subsp. avium, M. avium subsp. paratuberculosis-infected cells showed greater expression of IL-10 (6 and 24 h) and less expression of TNF-alpha (6 h). Therefore, the combination of inherent resistance to intracellular degradation and suppression of macrophage activation through oversecretion of IL-10 may contribute to the virulence of M. avium subsp. paratuberculosis in cattle.