结核分枝杆菌与巨噬细胞相互作用的研究进展

结核分枝杆菌是一种胞内感染菌,巨噬细胞是其寄生场所。结核分枝杆菌通过阻止吞噬溶酶体的融合、减少巨噬细胞凋亡、降低巨噬细胞对刺激应答的敏感性等途径逃避巨噬细胞的免疫监视和攻击,并在细胞内存活、增殖;而巨噬细胞又是抗菌免疫的主要效应细胞,通过直接杀伤和分泌多种细胞因子,对结核分枝杆菌具有免疫调节、呈递抗原等作用。深入研究结核分枝杆菌对巨噬细胞的免疫逃逸机制及巨噬细胞抗结核免疫作用,对研究宿主抗结核免疫机制及设计新型结核病疫苗有重要意义。     

寡核苷酸芯片研究结核分枝杆菌临床株和无毒株诱导的巨噬细胞U937凋亡相关基因的差异表达

结核病仍然是人类健康的主要威胁,结核分枝杆菌诱导的巨噬细胞凋亡是宿主防御反应之一,研究凋亡相关基因的差异表达有助于认识结核分枝杆菌致病机理和发现新的药物靶标。利用包括19200个基因或基因片段的DNA芯片研究巨噬细胞株U937对临床和实验室菌株感染的差异表达,Northem blotting和RT—PCR验证了芯片研究结果。Mtb H37Rv感染下调bcl—2,vitaminD受体、干扰素调控因子3、细胞色素氧化酶C表达,幅度分别为2-,3-,3-,2．5-倍,临床菌株感染上调SOD2、SOD3、丝氨酸蛋白酶、toll—like受体2、signal transducer and activator(STAT1)、hypoxia—inducible factor22等表达,幅度分别为2．9-,2．5-,2．5-,22-,2．4-,5．9-倍。结果提示,临床菌株感染更多促进凋亡,限制宿主的杀灭机理。该研究为进一步研究导致这些差异表达的结核分枝杆菌成分提供了基础。     

检测不同毒力结核分枝杆菌感染对巨噬细胞凋亡的影响及其Caspase-3和Bcl-2的表达

该文为探讨不同毒力的结核分枝杆菌感染对巨噬细胞凋亡的调控作用及其机制。实验用结核分枝杆菌国际标准强毒株H37Rv株和卡介苗BCG分别感染巨噬细胞RAW264.7株,同时设空白对照组,在感染后1,6,12,24 h,用流式细胞技术检测各组巨噬细胞的凋亡率,应用Western blot检测细胞Caspase-3和Bcl-2蛋白表达。结果发现,结核分枝杆菌感染组的凋亡率显著高于对照组,差异具有统计学意义(P<0.05);BCG感染组凋亡率高于H37Rv感染组,在感染后1,12,24 h凋亡率显著升高,差异具有统计学意义(P<0.05)。巨噬细胞感染结核分枝杆菌后其Caspase-3蛋白表达增高,结核分枝杆菌感染组的Caspase-3蛋白表达高于对照组:对照组相似文献   

自噬在结核分枝杆菌感染中作用的研究进展

结核病是一种由结核分枝杆菌感染引发的全球致死性传染病。结核分枝杆菌感染巨噬细胞后其在胞内的命运与细胞自噬的发生息息相关。但是,由于结核分枝杆菌与细胞自噬之间的博弈过程十分复杂且机制尚未完全阐明。因此,该文以结核分枝杆菌感染巨噬细胞后经典自噬和LAP对结核病发生发展的作用为切入点,从自噬抗结核分枝杆菌感染与结核分枝杆菌破坏自噬发生两个方面阐述了自噬与结核分枝杆菌之间的相互作用,从而为结核病发病机制的研究提供新的思路和视角。     

结核分枝杆菌Rv1057基因对巨噬细胞感染早期细胞因子表达的影响分析

Rv1057是结核分枝杆菌中唯一的7-折叠片β-螺旋蛋白,其生物学功能尚不清楚。为探讨Rv1057基因在结核分枝杆菌感染初期对巨噬细胞免疫应答的影响,利用Rv1057基因缺失菌株D1057和野生型H37Rv菌株感染巨噬细胞,检测结核分枝杆菌在巨噬细胞内的增殖速度,分析巨噬细胞在感染初期的细胞因子表达量变化。研究结果表明:D1057菌株在巨噬细胞内的活菌数量和增殖速度都显著低于H37Rv,被D1057感染的巨噬细胞中IL-1β、IL-10、TNF-α和IFN-γ表达量显著降低,但IL-8大量表达且无显著差异。上述研究结果证实,缺失Rv1057会降低结核分枝杆菌刺激巨噬细胞产生某些细胞因子的能力,明确了Rv1057基因参与结核分枝杆菌与巨噬细胞之间的免疫应答,为进一步解析Rv1057基因的生物学功能、结核分枝杆菌的致病机制奠定了基础。     

结核分枝杆菌酸抗性基因及其调控网络

病原菌在宿主细胞内的持留分子机理是目前研究的热点和难点。病原菌的抗酸能力与此密切相关。结核分枝杆菌感染导致的结核病仍然是全球公共卫生的重大威胁,这与结核分枝杆菌抗酸并在宿主巨噬细胞内持留有关。结核分枝杆菌抗酸主要通过调控质子进出、代谢调控胞内酸碱平衡和双组份信号系统调控。本文综述了结核分枝杆菌在酸胁迫下的整体调控网络,阐述了在酸性环境中结核分枝杆菌的具体调控机理,旨在为持留结核分枝杆菌的治疗提供新的全局性思路,寻找新的结核病防控靶标。     

HBHA 在结核病研究中的应用*

结核分枝杆菌hbhA编码基因是目前发现的与肺外结核转移相关的基因,其表达产物结核分枝杆菌肝素结合血凝黏附素(HBHA),是结核分枝杆菌表面表达和分泌的一种糖蛋白。HBHA具有介导肺结核和肺外结核的发生,参与实现MTB逃逸吞噬体进入细胞质中生存和繁殖,引起巨噬细胞凋亡等重要生物学功能,同时HBHA作为特异性抗原,用作免疫学检测具有较好的效果,并且动物实验证实HBHA具有明确的免疫治疗作用。因此,HBHA在结核病的免疫学诊断及免疫治疗方面,具有广阔的应用前景。     

结核分枝杆菌持续感染相关基因及其调控网络分析

结核分枝杆菌是结核病的致病菌, 也是迄今最成功的人类致病菌之一. 结核分枝杆菌能逃避宿主免疫攻击, 在人体内持续感染或呈休眠状态. 当人体免疫功能低下时, 持续感染或休眠的致病菌可能被重新激活. 结核分枝杆菌的持续感染是制约结核病控制计划成功的主要障碍之一. 揭示结核分枝杆菌持续感染的分子机制、寻找其中薄弱环节、发现适当的药物靶标并开发全新药物及免疫干预措施, 被认为是遏制结核病蔓延的关键. 结核分枝杆菌持续感染和再激活是众多基因协同的系统适应过程. 本文在全面分析全球结核分枝杆菌持续感染相关基因研究文献的基础上, 通过文本挖掘, 综合本实验室前期研究结果, 提出了结核分枝杆菌持续感染相关基因的调控网络, 为揭示结核分枝杆菌持续感染的机制, 筛选控制结核病的新靶标和免疫干预节点提供研究基础.     

结核病人感染人类免疫缺陷病毒导致血浆中和结核分枝杆菌诱生的细胞因子变化

在全球感染性疾病中,结核病仍是导致死亡的最普遍的原因之。人类免疫缺陷病毒(HIV)感染与结核病有着密切联系,它能加剧结核病的发展,结核病又可促进HIV的复制,加速艾滋病的发作。机体对结核分枝杆菌(简称结核杆菌)的抵抗主要依赖细胞免疫,T细胞产生的细胞因子与结核杆菌感染密切相关。Th1细胞活化分泌IFN—r,激活巨噬细胞对控制感染起着重要作用。     

耐药结核分枝杆菌潜伏-复发感染动物模型的研究进展

潜伏结核感染(latent tuberculosis infection,LTBI)复发是新发结核病的主要来源,其中耐药结核病所占比例较大,使耐药LTBI复发的防控成为结核病研究的重点。耐药结核分枝杆菌潜伏-复发感染动物模型是开展耐药结核病防控相关机制研究、抗耐药结核分枝杆菌药物和疫苗研究的基础。目前耐药结核分枝杆菌感染动物模型缺乏,而已有的结核分枝杆菌标准株H37Rv潜伏-复发感染模型存在缺陷,如小鼠模型的潜伏期荷菌量偏高、复发期变异大,而猴模型的潜伏期和复发期不可预测。模型的可控性差使其应用困难,且缺乏可用的免疫学评价指标,导致远期复发无法预测。因此,基于现有H37Rv潜伏-复发感染动物模型的制备方法,展望耐药结核分枝杆菌潜伏-复发感染动物模型可能存在的缺陷,通过选用新的抑菌剂和诱导剂,制备有稳定潜伏期、潜伏时长适中、复发起点和复发水平变异小的动物模型,是未来耐药结核分枝杆菌潜伏-复发感染动物模型研究的方向。     

Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophages

Human alveolar macrophages (AMphi) undergo apoptosis following infection with Mycobacterium tuberculosis in vitro. Apoptosis of cells infected with intracellular pathogens may benefit the host by eliminating a supportive environment for bacterial growth. The present study compared AMphi apoptosis following infection by M. tuberculosis complex strains of differing virulence and by Mycobacterium kansasii. Avirulent or attenuated bacilli (M. tuberculosis H37Ra, Mycobacterium bovis bacillus Calmette-Guérin, and M. kansasii) induced significantly more AMphi apoptosis than virulent strains (M. tuberculosis H37Rv, Erdman, M. tuberculosis clinical isolate BMC 96.1, and M. bovis wild type). Increased apoptosis was not due to greater intracellular bacterial replication because virulent strains grew more rapidly in AMphi than attenuated strains despite causing less apoptosis. These findings suggest the existence of mycobacterial virulence determinants that modulate the apoptotic response of AMphi to intracellular infection and support the hypothesis that macrophage apoptosis contributes to innate host defense in tuberculosis.     

Macrophage apoptosis in response to high intracellular burden of Mycobacterium tuberculosis is mediated by a novel caspase-independent pathway

We previously reported that macrophage exposure to attenuated strains of pathogenic mycobacteria at multiplicities of infection (MOI) < or = 10 triggers TNF-alpha-mediated apoptosis which reduces the viability of intracellular bacilli. Virulent strains were found to suppress macrophage apoptosis, and it was proposed that apoptosis is an innate defense against intracellular Mycobacterium tuberculosis analogous to apoptosis of virus-infected cells. The potential similarity of host cell responses to intracellular infection with mycobacteria and viruses suggests that M. tuberculosis might lyse infected macrophage when that niche is no longer needed. To investigate this question, we challenged murine macrophages with high intracellular bacillary loads. A sharp increase in cytolysis within 24 h was observed at MOI > or = 25. The primary death mode was apoptosis, based on nuclear morphology and phosphatidyl serine exposure, although the apoptotic cells progressed rapidly to necrosis. Apoptosis at high MOI differs markedly from low MOI apoptosis: it is potently induced by virulent M. tuberculosis, it is TNF-alpha-independent, and it does not reduce mycobacterial viability. Caspase inhibitors failed to prevent high MOI apoptosis, and macrophages deficient in caspase-3, MyD88, or TLR4 were equally susceptible as wild type. Apoptosis was reduced in the presence of cathepsin inhibitors, suggesting the involvement of lysosomal proteases in this novel death response. We conclude that the presence of high numbers of intracellular M. tuberculosis bacilli triggers a macrophage cell death pathway that could promote extracellular spread of infection and contribute to the formation of necrotic lesions in tuberculosis.     

Effect of proton pump inhibitor alone or in combination with clarithromycin on mycobacterial growth in human alveolar macrophages

The effect of omeprazole, a clinically used proton pump inhibitor, alone or in combination with clarithromycin was evaluated against Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium tuberculosis, using a human alveolar macrophage model of infection. Omeprazole exhibited no significant effect on the growth of the two M. avium complex strains or on the mycobactericidal activity of clarithromycin against them. In contrast, omeprazole significantly promoted the growth of Mycobacterium tuberculosis and the anti-mycobacterial activity of clarithromycin against it in human alveolar macrophages. It was speculated that intracellular acidic milieu around M. tuberculosis might be one reason for the lower activity of clarithromycin in the treatment of human tuberculosis.     

Induction of TNF in human alveolar macrophages as a potential evasion mechanism of virulent Mycobacterium tuberculosis.

The ability of macrophages to release cytokines is crucial to the host response to intracellular infection. In particular, macrophage-derived TNF plays an important role in the host response to infection with the intracellular pathogen Mycobacterium tuberculosis. In mice, TNF is indispensable for the formation of tuberculous granulomas, which serve to demarcate the virulent bacterium. TNF is also implicated in many of the immunopathological features of tuberculosis. To investigate the role of TNF in the local immune response, we infected human alveolar macrophages with virulent and attenuated mycobacteria. Infection with virulent strains induced the secretion of significantly higher levels of bioactive TNF than attenuated strains correlating with their ability to multiply intracellularly. Treatment of infected macrophages with neutralizing anti-TNF Abs reduced the growth rate of intracellular bacteria, whereas bacterial replication was augmented by addition of exogenous TNF. Infected and uninfected macrophages contributed to cytokine production as determined by double-staining of M. tuberculosis and intracellular TNF. The induction of TNF by human alveolar macrophages at the site of infection permits the multiplication of intracellular bacteria and may therefore present an evasion mechanism of human pathogens.     

HIV impairs TNF-alpha mediated macrophage apoptotic response to Mycobacterium tuberculosis

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV(+) persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-alpha. In contrast, apoptosis of differentiated HIV(+) human U1 macrophages (HIV(+) U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-alpha release, whereas apoptosis and TNF-alpha release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-alpha release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-kappaB nuclear translocation were reduced in HIV(+) U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIV(+) persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-alpha release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIV(+) persons.     

CD43 controls the intracellular growth of Mycobacterium tuberculosis through the induction of TNF-alpha-mediated apoptosis

Establishment of Tuberculosis infection begins with the successful entry and survival of the pathogen within macrophages. We previously showed that macrophage CD43 is required for optimal uptake and growth inhibition of Mycobacterium tuberculosis both in vitro and in vivo. Here, we explore the mechanisms by which CD43 restricts mycobacterial growth in murine macrophages. We found that although M. tuberculosis grows more readily in resting CD43-/- macrophages, priming of cells with IFN-gamma returns the bacterial growth rate to that seen in CD43+/+ cells. To discern the mechanisms by which M. tuberculosis exhibits enhanced growth within resting CD43-/- macrophages, we assessed the induction of inflammatory mediators in response to infection. We found that absence of CD43 resulted in reduced production of TNF-alpha, IL-12 and IL-6 by M. tuberculosis-infected macrophages. We also found that infected resting, but not activated CD43-/- macrophages, showed decreased apoptosis and increased necrosis. Exogenous addition of the pro-inflammatory cytokine TNF-alpha restored control of M. tuberculosis growth and induction of apoptosis to CD43+/+ levels. We propose that CD43 is involved in the inflammatory response to M. tuberculosis and, through the induction of pro-inflammatory mediators, can regulate apoptosis to control intracellular growth of the bacterium.     

Activation of JAK2/STAT1-alpha-dependent signaling events during Mycobacterium tuberculosis-induced macrophage apoptosis

Induction of apoptosis by Mycobacterium tuberculosis in murine macrophage involves TNF-alpha and nitric oxide (NO) production and caspase cascade activation; however, the intracellular signaling pathways implicated remain to be established. Our results indicate that infection of the B10R murine macrophage line with M. tuberculosis induces apoptosis independent of mycobacterial phagocytosis and that M. tuberculosis induces protein tyrosine kinase (PTK) activity, JAK2/STAT1-alpha phosphorylation, and STAT1-alpha nuclear translocation. Inhibitors of PTK (AG-126), or JAK2 (AG-490) inhibited TNF-alpha and NO production, caspase 1 activation and apoptosis, suggesting that M. tuberculosis-induction of these events depends on JAK2/STAT1-alpha activation. In addition, we have obtained evidence that ManLAM capacity to inhibit M. tuberculosis-induced apoptosis involves the activation of the PTP SHP-1. The finding that M. tuberculosis infection activate JAK2/STAT1-alpha pathway suggests that M. tuberculosis might mimic macrophage-activating stimuli.     

Protein kinase E of Mycobacterium tuberculosis has a role in the nitric oxide stress response and apoptosis in a human macrophage model of infection

Mycobacterium tuberculosis , an intracellular pathogen, inhibits macrophage apoptosis to support survival and replication inside the host cell. We provide evidence that the functional serine/threonine kinase, PknE, is important for survival of M. tuberculosis that enhances macrophage viability by inhibiting apoptosis. A promoter of PknE identified in this study was shown to respond to nitric oxide stress. Deletion of pknE in virulent M. tuberculosis , H37Rv, resulted in a strain that has increased resistance to nitric oxide donors and increased sensitivity to reducing agents. The deletion mutant created by specialized transduction induced enhanced apoptosis while inhibiting necrosis. The pknE mutant also modifies the innate immune response as shown by the marked decline in the pro-inflammatory cytokines in a macrophage model of infection. These findings suggest a novel mechanism, by which PknE senses nitric oxide stress and prevents apoptosis by interfering with host signalling pathways.     

结核分枝杆菌对宿主巨噬细胞死亡方式的调控

结核分枝杆菌（Mycobacterium tuberculosis,Mtb）感染后能抑制宿主巨噬细胞（M西）的免疫反应,并在其中生存、复制。研究表明Mtb减毒株感染主要诱导宿主Mφ凋亡,凋亡能抑制胞内Mtb的活力;而Mtb毒力株感染能抑制凋亡的完成,诱导Mφ坏死,最终导致Mtb扩散、感染临近细胞。通过对Mtb感染诱导宿主Mφ不同死亡方式的讨论,进一步认识Mtb的致病机制。