不同毒力结核分枝杆菌感染对巨噬细胞铁蛋白及铁转运蛋白表达的影响

目的探讨结核分枝杆菌国际标准强毒株H37Rv株(简称H37Rv株)和卡介苗菌株(简称BCG菌株)分别感染巨噬细胞后,各感染组巨噬细胞内铁蛋白(Fn)和铁转运蛋白(FPN)表达量及其时相性变化。方法分别用结核分枝杆菌H37Rv株和BCG菌株感染巨噬细胞RAW264.7细胞株,于感染后1、6、12、18、24 h,应用ELISA检测各组感染巨噬细胞培养上清液中Fn和FPN的含量;应用Western blot技术检测上述时间点各组感染巨噬细胞内Fn的表达量。结果不同菌株感染巨噬细胞后,巨噬细胞内Fn随处理时间延长表达逐渐增强;感染1 h,正常对照组Fn的表达高于H37Rv组和BCG组;感染6 h,感染组表达高于正常对照组,感染12 h,H37Rv组高于正常对照组,差异具有统计学意义(P<0.05);感染18 h和24 h,H37Rv组>BCG组>正常对照组,差异具有统计学意义(P<0.05)。FPN的表达量随时间变化逐渐降低。与正常对照组相比,H37Rv组、BCG组FPN的表达均呈现较低水平,为H37Rv组相似文献   

LprG and PE_PGRS33 Mycobacterium tuberculosis virulence factors induce differential mitochondrial dynamics in macrophages

The interaction of a pathogen with its host cell takes place at different levels, including the bioenergetics adaptation of both the pathogen and the host cell in the course of an infection. In this regard, Mycobacterium tuberculosis infection of macrophages induces mitochondrial membrane potential (Δψm) changes and cytochrome c release, depending on the bacteria strain's virulence, and the mitochondrial dynamics is modified by pathogens, such as Listeria monocytogenes. Here, we investigated whether two M. tuberculosis virulence factors are able to induce distinguishable bioenergetics traits in human monocyte‐derived macrophages (MDMs). Results showed that Rv1411c (LprG, p27) induced mitochondrial fission, lowered the cell respiratory rate and modified the kinetics of mitochondrial Ca²⁺ uptake in response to agonist stimulation. In contrast, Rv1818c (PE_PGRS33) induced mitochondrial fusion, but failed to induce any appreciable effect on cell respiratory rate or mitochondrial Ca²⁺ uptake. Overall, these results suggest that two different virulence factors from the same pathogen (M. tuberculosis) induce differential effects on mitochondrial dynamics, cell respiration and mitochondrial Ca²⁺ uptake in MDMs. The timing of differential mitochondrial activity could ultimately determine the outcome of host‐pathogen interactions.     

Leishmania major-mediated prevention of programmed cell death induction in infected macrophages is associated with the repression of mitochondrial release of cytochrome c

Leishmania are obligate, intracellular parasites of macrophages in their vertebrate hosts, including humans, in which they cause disease. Here, we report that in vitro infection with Leishmania major protects murine bone marrow-derived macrophages against programmed cell death (PCD) induced by deprival of macrophage-colony stimulating factor and delays PCD caused by treatment with staurosporine, a broad inducer of PCD. This preventive effect was observed in macrophages from L. major-susceptible BALB/c and L. major-resistant C57BL/6 mice, indicating that repression of PCD did not depend on genetic background-specific regulation of T helper cell type 1 (Th1)/Th2 cytokine secretion. Prevention of effector caspase activation and PCD was associated with a repression of mitochondrial release of cytochrome c and did not involve the nuclear factor-kappaB pathway. The capacity of L. major to delay PCD induction in the infected macrophages may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.     

靶向沉默Mcl-1基因对感染不同毒力结核杆菌小鼠腹腔巨噬细胞凋亡的影响

 目的: 通过RNA干扰技术特异性沉默 Mcl-1 基因,探讨下调 Mcl-1 基因对感染不同毒力结核杆菌小鼠腹腔巨噬细胞凋亡的影响。方法: 分别用制备好的新疆地区流行的优势强毒结核分枝杆菌临床分离株(简称强毒株)、结核分枝杆菌国际标准强毒株H37Rv(简称H37Rv)、结核分枝杆菌国际标准无毒株H37Ra(简称H37Ra)和卡介苗(BCG)菌悬液感染BALB/c小鼠,再以筛选并构建好的Mcl-1-shRNA作用于感染的小鼠模型,同时设立相应对照组,于作用后1 d、3 d、5 d和7 d提取小鼠腹腔巨噬细胞,应用实时荧光定量PCR和Western blot检测各组小鼠腹腔巨噬细胞中Mcl-1 mRNA和蛋白的表达;应用流式细胞术检测各组巨噬细胞的凋亡水平。结果: 小鼠被不同毒力的结核杆菌感染后其腹腔巨噬细胞中Mcl-1 mRNA和蛋白的表达水平均有不同程度的升高,其中以感染了强毒株和H37Rv的腹腔巨噬细胞升高最为明显(P<0.05);应用RNA干扰技术沉默 Mcl-1 基因后,Mcl-1 mRNA和蛋白的表达水平明显低于对照组(P<0.05);流式细胞术分析显示,下调 Mcl-1 基因的表达可诱导小鼠腹腔巨噬细胞凋亡。结论: 应用Mcl-1-shRNA可有效沉默 Mcl-1 在感染了不同毒力结核杆菌小鼠腹腔巨噬细胞中的表达,并能上调巨噬细胞的凋亡水平。     

结核杆菌H37Ra感染巨噬细胞的研究

目的:研究结核杆菌H37Ra感染巨噬细胞后氮氧化物的产生及细胞因子的分泌和表达情况.方法:结核杆菌H37Ra感染RAW264.7巨噬细胞株24h后收集上清液,用Griess法检测一氧化氮(NO),化学法检测过氧化氢(H2O2)的产生,逆转录-聚合酶链反应(RT-PCR)法检测感染后巨噬细胞IL-12和TNF-α mRNA的表达,ELISA法检测细胞因子IL-12和TNF-α的含量.结果:结核杆菌H37Ra感染巨噬细胞后产生的NO和H2 O2水平均显著升高,同时IL-12和TNF-α的分泌和表达也明显增加(P＜0.05).结论:结核杆菌H37Ra感染巨噬细胞后能产生大量的氮氧化物和抗结核细胞因子,从而产生有利于宿主的抗结核免疫应答.     

Mycobacterium tuberculosis blocks crosslinking of annexin-1 and apoptotic envelope formation on infected macrophages to maintain virulence

Macrophages infected with attenuated Mycobacterium tuberculosis strain H37Ra become apoptotic, which limits bacterial replication and facilitates antigen presentation. Here we demonstrate that cells infected with H37Ra became apoptotic after the formation of an apoptotic envelope on their surface was complete. This process required exposure of phosphatidylserine on the cell surface, followed by deposition of the phospholipid-binding protein annexin-1 and then transglutaminase-mediated crosslinking of annexin-1 through its amino-terminal domain. In macrophages infected with the virulent strain H37Rv, in contrast, the amino-terminal domain of annexin-1 was removed by proteolysis, thus preventing completion of the apoptotic envelope, which resulted in macrophage death by necrosis. Virulent M. tuberculosis therefore avoids the host defense system by blocking formation of the apoptotic envelope, which leads to macrophage necrosis and dissemination of infection in the lung.     

Kinetics of chemokine secretion in human macrophages infected with various strains of Mycobacterium tuberculosis

Background and Purpose: It has been shown that chemokine secretion upon infection with Mycobacterium tuberculosis is influenced by the virulence of the strain, and it is suggested that virulence-associated differences in chemokine secretion contribute to the failure in containing the infection due to poor granuloma formation. Materials and Methods: In this study, we used prevalent M tuberculosis clinical strains (S7 and S10) to study the chemokine secretion profile in infected THP-1 cells and monocyte-derived macrophages (MDM) and compared this with the chemokine secretion induced by laboratory strains. Results: This study showed that comparatively lower levels of IP-10 were induced by clinical strains than by laboratory strains in both differentiated THP-1 and MDMs. The secretion of MIP-1α was also depressed but only in the THP-1 cells infected with clinical strains. This depressed chemokine secretion may hinder the movement of Th-1 cells from the periphery into the infection foci to control the infection. Correlation between IP-10 and IL-12p40 showed a negative relationship in control MDMs, while there was a positive correlation in all the infected strains, indicating their cooperative role in attracting and activating Th1 cells for a protective immune response at the site. This relationship was strain dependent, with avirulent H37Ra showing higher correlation, followed by the clinical strains and the virulent H37Rv. A positive correlation of IP-10 with IFN-γ (S7 and H37Ra) and with IL-10 (H37Ra and H37Rv) suggested a definitive interplay of these molecules in infection. Conclusions: The chemokines secretion by infected THP-1 cells and MDMs was strain dependant and the lower induction by the clinical strains may indicate that the clinical strains maintain a quiescent nature to mislead the host immune system for their benefit.     

Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes

It has been shown recently that different genotypes of Mycobacterium tuberculosis induce distinct immune responses in the host, as reflected by variations in cytokine and iNOS expression. Because these molecules are probably regulated by multiple factors in vivo this complex phenomenon was partially analysed by assessing cytokine and iNOS expression by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in an in vitro model of bone marrow-derived macrophages infected with three different M. tuberculosis genotypes: Canetti, H37 Rv and Beijing. Although the three genotypes induced production of iNOS and the different cytokines tested at 24 h post-infection, macrophages infected with the Beijing isolate expressed the highest levels of mRNA for iNOS, interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, IL-12 cytokines and lower levels of IL-10 compared with cells infected with other genotypes. This expression pattern has been associated with infection control, but during infection in vivo with the Beijing genotype it is lost upon progression to chronic phase. The failure to control infection is likely to be influenced by cytokines produced by other cell types and bacterial molecules expressed during the course of disease. Results presented in this work show that each genotype has the ability to induce different levels of cytokine expression that could be related to its pathogenesis during infection.     

Interleukin-10 production by lung macrophages in CBA xid mutant mice infected with Mycobacterium tuberculosis

Mice on the CBA inbred strain background expressing the well characterized mutation designated xid in the cytoplasmic signalling enzyme Bruton's protein kinase have been previously noted to illustrate shifts in T helper type 1 (Th1)/Th2 immunity which is underlined by an apparent failure to produce the regulatory cytokine interleukin-10. In the current study we examined if this extended to infection with Mycobacterium tuberculosis, which also depends on Th1 immunity. Contrary to expectations, xid mice showed evidence of a transient early susceptibility to pulmonary infection, changes in macrophage morphology, and decreased activation of lung natural killer cells, while showing evidence of substantial IL-10 production and accumulation in lung lesions macrophages, but paradoxically this did not influence the course of the chronic disease. In addition, macrophages from the lungs of xid mice also expressed high levels of CD14. These observations suggest that the xid mutation in cellular signalling has much wider effects on the immune system than previously thought.     

The effect of iron on the expression of cytokines in macrophages infected with Mycobacterium tuberculosis

Iron is known to play an important role in different bacterial infections and, in particular, in their development. One example is infection with Mycobacterium tuberculosis where iron contributes to growth and survival of the bacteria within the host cell. The majority of studies performed on tuberculosis have focused on the direct effect of iron on bacterial growth; however, little is known about how iron modifies the mycobacterial-host interaction. In order to address this, we have investigated the effect of iron on intracellular growth of M. tuberculosis in J774 macrophages and the molecular mechanisms that are affected during this interaction. We observed that iron modifies intracellular growth of the mycobacteria and that their growth kinetics was modified from that observed for the extracellular situation in the presence of iron. Similarly, when iron was present during the infection, there was a reduced release of tumour necrosis factor-alpha and it was related to a higher number of bacilli inside the host cell and low expression of interleukin-1 (IL-1) and IL-6 mRNA. Hence, this work demonstrates that iron, besides promoting mycobacterial growth, also regulates the relationship between macrophage and bacteria.     

Molecular characterization of clinical isolates of Mycobacterium tuberculosis and their association with phenotypic virulence in human macrophages

Among 125 clinical isolates of Mycobacterium tuberculosis collected in Hong Kong and Shanghai, China, between 2002 and 2004, IS6110 typing revealed that 71 strains (57%) belonged to the Beijing family. The intracellular growth of the strains in human peripheral blood monocyte-derived macrophages was measured ex vivo on days 0, 3, 6, and 10. Among all tested strains, three hypervirulent strains showed significant increases in intracellular growth after 10 days of incubation. With an initial bacterial load of 10(4) CFU, most of the clinical isolates and H37Ra (an avirulent strain) exhibited no intracellular survival on day 10, while the three hypervirulent strains together with H37Rv (a virulent strain) showed on average a two- to fourfold rise in CFU count. These three hypervirulent strains belonging to a non-Beijing family were isolated from patients suffering from tuberculosis meningitis. Cytokines secreted by gamma interferon-activated macrophages were measured daily after challenge with selected strains of M. tuberculosis. The levels of tumor necrosis factor alpha were elevated after 24 h of infection among all strains, but the levels were significantly lower among the three hypervirulent strains, whereas interleukin 10 (IL-10) and IL-12 were not detected. Results were concordant with the differential expression of the corresponding cytokine genes in activated macrophages, as monitored by real-time PCR. Our findings highlighted that these three hypervirulent strains may possess an innate mechanism for escaping host immunity, which accounts for their characteristic virulence in patients presenting with a more severe form of disease.     

Parkin prevents mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death

Parkin gene mutations have been implicated in autosomal-recessive early-onset parkinsonism and lead to specific degeneration of dopaminergic neurons in midbrain. To investigate the role of Parkin in neuronal cell death, we overproduced this protein in PC12 cells in an inducible manner. In this cell line, neuronally differentiated by nerve growth factor, Parkin overproduction protected against cell death mediated by ceramide, but not by a variety of other cell death inducers (H(2)O(2), 4-hydroxynonenal, rotenone, 6-OHDA, tunicamycin, 2-mercaptoethanol and staurosporine). Protection was abrogated by the proteasome inhibitor epoxomicin and disease-causing variants, indicating that it was mediated by the E3 ubiquitin ligase activity of Parkin. Interestingly, Parkin acted by delaying mitochondrial swelling and subsequent cytochrome c release and caspase-3 activation observed in ceramide-mediated cell death. Subcellular fractionation demonstrated enrichment of Parkin in the mitochondrial fraction and its association with the outer mitochondrial membrane. Together, these results suggest that Parkin may promote the degradation of substrates localized in mitochondria and involved in the late mitochondrial phase of ceramide-mediated cell death. Loss of this function may underlie the degeneration of nigral dopaminergic neurons in patients with Parkin mutations.     

Chemokine response in mice infected with Mycobacterium tuberculosis.

We show here that infection of murine macrophages with various strains of Mycobacterium tuberculosis induces the rapid in vitro expression of genes encoding chemokines macrophage inflammatory protein 1 alpha and macrophage inflammatory protein 2, which recruit neutrophils to sites of infection, and macrophage-recruiting chemokines 10-kDa, interferon-inducible protein (IP-10) and macrophage chemotactic protein 1. Three strains of M. tuberculosis, Erdman and the clinical isolates CSU 22 and CSU 46, induced similar levels of secretion of macrophage chemotactic protein 1 from infected macrophage monolayers; however, the Erdman strain failed to induce levels of secretion of tumor necrosis factor alpha similar to those induced by either CSU 22 or CSU 46. Using a low-dose aerosol infection model, we also found that while the Erdman strain induced negligible increases in chemokine mRNA levels in the lungs, infection with either CSU 22 or CSU 46 resulted in greater levels of mRNA production for all four chemokines tested. The growth of these strains in the lungs was, however, equally well contained by acquired host immunity. These data allow us to hypothesize that the chemokine response in the lungs probably does not control the protective granulomatous response and that perhaps other T-cell- or macrophage-associated cytokines such as tumor necrosis factor alpha or interleukin 12 may be involved in this process.     

T-Cell hyporesponsiveness induced by activated macrophages through nitric oxide production in mice infected with Mycobacterium tuberculosis.

In active tuberculosis, T-cell response to Mycobacterium tuberculosis is known to be reduced. In the course of Mycobacterium tuberculosis infection in mice, we observed that T-cell proliferation in response to M. tuberculosis purified protein derivative (PPD) reached the maximum level on day 7, then declined to the minimal level on day 14, and persisted at a low level through day 28 postinfection. The frequency of PPD-specific CD4 T cells in the spleen on day 28 decreased to one-sixth on day 7. To further investigate the mechanism of this T-cell hyporesponsiveness, we next analyzed the suppressive activity of spleen macrophages on T-cell function. The nonspecific proliferative response of naive T cells and the PPD-specific proliferative response of T cells were suppressed by day 28 macrophages, but not by day 7 macrophages or naive macrophages. This reduction of proliferative response was restored by addition of nitric oxide synthesis inhibitor, NG-monoethyl-L-arginine monoacetate, but not by monoclonal antibody against interleukin 10 or transforming growth factor beta. These data indicate that the macrophages from mice chronically infected with M. tuberculosis suppress T-cell response through production of nitric oxide, suggesting that nitric oxide-induced elimination mediated by activated macrophages may reduce the T-cell response and the number of mycobacterium-specific CD4 T cells in vivo.     

Mycobacterium bovis BCG vaccination augments interleukin-8 mRNA expression and protein production in guinea pig alveolar macrophages infected with Mycobacterium tuberculosis

Alveolar macrophages are likely the first cell type to encounter Mycobacterium tuberculosis in a pulmonary infection, resulting in the production of chemokines. In order to evaluate this response, alveolar macrophages harvested from nonvaccinated and Mycobacterium bovis BCG-vaccinated guinea pigs were infected in vitro with live M. tuberculosis H37Ra or H37Rv (multiplicity of infection, 1:1) or cultured with lipopolysaccharide (10 micro g/ml) for 3, 12, and 24 h. Interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) mRNA expression was determined by real-time PCR. Culture supernatants were assayed for guinea pig IL-8 protein by using a human IL-8 enzyme-linked immunosorbent assay kit. Alveolar macrophages harvested from BCG-vaccinated guinea pigs produced significantly more mRNA and protein for IL-8 than alveolar macrophages harvested from nonvaccinated guinea pigs at 12 and 24 h poststimulation or postinfection. Infection with attenuated M. tuberculosis (H37Ra) stimulated alveolar macrophages isolated from BCG-vaccinated guinea pigs to produce significantly more IL-8 mRNA than did alveolar macrophages infected with a virulent strain (H37Rv) at 12 and 24 h postinfection. Significant MCP-1 mRNA production was also detected in stimulated or infected alveolar macrophages; however, prior vaccination did not significantly affect levels of MCP-1 mRNA. Alveolar macrophages isolated from BCG-vaccinated guinea pigs produced significantly more IL-8 mRNA and protein when stimulated for 24 h with heat-killed H37Ra, heat-killed H37Rv, and H37Rv cell wall, but not mannose-capped lipoarabinomannan (ManLAM), than did cells stimulated with media alone. These observations indicate that prior vaccination may alter very early events in the M. tuberculosis-infected lung.     

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.     

Pathogenesis of tuberculosis: interaction of Mycobacterium tuberculosis with macrophages.

Central to understanding the pathogenesis of tuberculosis is the interaction between the pathogen and mononuclear phagocytes. A key question about that interaction is whether Mycobacterium tuberculosis exerts an effect on phagolysosome fusion. We have reexamined the dynamics of phagolysosome fusion and its effect on intracellular bacterial replication in M. tuberculosis-infected macrophages by performing an extensive study at the electron microscopic level. Thoria-labelled murine and human macrophages were infected with a virulent (H37Rv) or avirulent (H37Ra) strain of M. tuberculosis or with Mycobacterium bovis BCG vaccine for times ranging from 2 h to 7 days. In all cases, by 2 h postinfection, approximately 85% of the bacteria clearly resided in fused vacuoles. However, at 4 days postinfection, fusion levels for viable H37Rv and H37Ra were reduced by half, whereas the fusion profiles of BCG and of heat-killed H37Rv and H37Ra were unchanged. A comparison of the numbers of bacteria per fused and nonfused vacuoles suggests both a net transfer of bacteria out of fused vacuoles and preferential bacterial multiplication in nonfused vacuoles. H37Rv and H37Ra appeared to bud from the phagolysosomes into tightly apposed membrane vesicles that did not fuse with secondary lysosomes. In some cases, no such membrane was seen and the bacteria appeared to be free in the cytoplasm. Only viable H37Rv showed a significant increase in bacterial counts during the course of infection. Thus, both of the attenuated strains we examined differed from the virulent strain H37Rv in their abilities to replicate successfully within macrophages, but each diverged from H37Rv at a different point in the process. Viable tubercle bacilli H37Rv and H37Ra had the capacity to escape from fused vesicles as the infection progressed; BCG did not. After extrusion from the phagolysosome, H37Rv, but not H37Ra, was able to multiply. These results suggest a novel mechanism by which virulent M. tuberculosis eludes the microbicidal mechanisms of macrophages by escaping from fused phagolysosomes into nonfused vesicles or the cytoplasm.