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（Mycobacterium tuberculosis,MTB）是一种典型的胞内致病菌,巨噬细胞是MTB在体内的主要宿主细胞。巨噬细胞具有强大的吞噬功能,在机体固有免疫和适应性免疫中均发挥着重要作用,可有效保护宿主免受结核分枝杆菌的感染。MTB在与宿主巨噬细胞的长期相互作用过程中,逐渐形成多种逃避杀灭的有效策略,得以在宿主体内存活并增殖。该文从巨噬细胞抗MTB感染及MTB逃避巨噬细胞杀灭两个方面综述国内外的研究进展。     

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。     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

Components derived from Pelargonium stimulate macrophage killing of Mycobacterium species

Aims:  To determine the capacity of extracts of Pelargonium reniforme and Pelargonium sidoides , plants of the Geraniaceae family, to stimulate the uptake and killing of mycobacteria by murine macrophages and to identify the constituents that are responsible.
Methods and results:  Bioassay-guided fractionation of aqueous P. reniforme extracts yielded five chemically distinct structures with the capacity to increase the rate of intracellular killing by macrophages. These were: gallic acid, methyl gallate, myricetin and quercitin-3- O -β- d -glucoside, in addition to the previously unrecognized constituent 1- O -(2-(4-methoxyphenyl)ethyl-6- O -galloyl-glucopyranoside. Kinetics of intracellular accumulation of Mycobacterium tuberculosis and Mycobacterium fortuitum by macrophages were indistinguishable; pure preparations of the four previously known plant constituents stimulated macrophage killing, but not uptake, of M. tuberculosis and M. fortuitum equally well.
Conclusions:  A number of distinct molecular species are present in the medicinal plant P. reniforme that stimulate the killing of the intracellular pathogen M. tuberculosis.
Significance and Impact of the Study:  These observations support the view that Pelargonium extracts may have utility in the treatment of tuberculosis.     

Rab GTPases regulating phagosome maturation are differentially recruited to mycobacterial phagosomes

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that can replicate within infected macrophages. The ability of M. tb to arrest phagosome maturation is believed to facilitate its intracellular multiplication. Rab GTPases regulate membrane trafficking, but details of how Rab GTPases regulate phagosome maturation and how M. tb modulates their localization during inhibiting phagolysosome biogenesis remain elusive. We compared the localization of 42 distinct Rab GTPases to phagosomes containing either Staphylococcus aureus or M. tb. The phagosomes containing S. aureus were associated with 22 Rab GTPases, but only 5 of these showed similar localization kinetics as the phagosomes containing M. tb. The Rab GTPases responsible for phagosome maturation, phagosomal acidification and recruitment of cathepsin D were examined in macrophages expressing the dominant-negative form of each Rab GTPase. LysoTracker staining and immunofluorescence microscopy revealed that Rab7, Rab20 and Rab39 regulated phagosomal acidification and Rab7, Rab20, Rab22b, Rab32, Rab34, Rab38 and Rab43 controlled the recruitment of cathepsin D to the phagosome. These results suggest that phagosome maturation is achieved by a series of interactions between Rab GTPases and phagosomes and that differential recruitment of these Rab GTPases, except for Rab22b and Rab43, to M. tb-containing phagosomes is involved in arresting phagosome maturation and inhibiting phagolysosome biogenesis.     

Differential recruitment of CD63 and Rab7‐interacting‐lysosomal‐protein to phagosomes containing Mycobacterium tuberculosis in macrophages

M.tb is an intracellular pathogen which survives within the phagosomes of host macrophages by inhibiting their fusion with lysosomes. Here, it has been demonstrated that a lysosomal glycoprotein, CD63, is recruited to the majority of M.tb phagosomes, while RILP shows limited localization. This is consistent with the author's findings that CD63, but not RILP, is recruited to the phagosomes in macrophages expressing the dominant negative form of Rab7. These results suggest that M.tb phagosomes selectively fuse with endosomes and lysosomes to escape killing activity while acquiring nutrients.     

Acridine Orange/exosomes increase the delivery and the effectiveness of Acridine Orange in human melanoma cells: A new prototype for theranostics of tumors

Specifically targeted drug delivery systems with low immunogenicity and toxicity are deemed to increase efficacy of cancer chemotherapy. Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466?nm. However, to date the clinical use of AO is limited by the potential side effects elicited by systemic administration. The endogenous nanocarrier exosomes have been recently introduced as a natural delivery system for therapeutic molecules. In this article, we show the outcome of the administration to human melanoma cells of AO charged Exosomes (Exo-AO), in both monolayer and spheroid models. The results showed an extended drug delivery time of Exo-AO to melanoma cells as compared to the free AO, improving the cytotoxicity of AO. This study shows that Exo-AO have a great potential for a real exploitation as a new theranostic approach against tumors based on AO delivered through the exosomes.     

Deletion of PIKfyve alters alveolar macrophage populations and exacerbates allergic inflammation in mice

Alveolar macrophages (AMs) are specialized tissue‐resident macrophages that orchestrate the immune responses to inhaled pathogens and maintain organ homeostasis of the lung. Dysregulation of AMs is associated with allergic inflammation and asthma. Here, we examined the role of a phosphoinositide kinase PIKfyve in AM development and function. Mice with conditionally deleted PIKfyve in macrophages have altered AM populations. PIKfyve deficiency results in a loss of AKT activation in response to GM‐CSF, a cytokine critical for AM development. Upon exposure to house dust mite extract, mutant mice display severe lung inflammation and allergic asthma accompanied by infiltration of eosinophils and lymphoid cells. Moreover, they have defects in production of retinoic acid and fail to support incorporation of Foxp3⁺ T_reg cells in the lung, resulting in exacerbation of lung inflammation. Thus, PIKfyve plays a role in preventing excessive lung inflammation through regulating AM function.     

结核分枝杆菌ESX-1分泌蛋白ESAT-6增强巨噬细胞吞噬功能

【目的】研究结核分枝杆菌(Mycobacterium tuberculosis)分泌蛋白ESAT-6(early secreted antigenictarget of 6 kDa)对巨噬细胞吞噬功能的影响。【方法】用重组质粒pFLAG-ESAT-6和pFLAG-EGFP转染RAW264.7细胞,经G418筛选,PCR、RT-PCR和Western blot鉴定,获得稳定表达flag-ESAT-6和flag-EGFP的RAW细胞系,然后用流式细胞术观察各稳转细胞系吞噬荧光微球的能力,并用共聚焦显微镜和菌落计数法检测稳转细胞系吞噬大肠杆菌(Escherichia coli)的能力。【结果】获得了稳定表达flag-ESAT-6的RAW-E6细胞系和表达flag-EGFP的RAW-EGFP细胞系;流式细胞术检测结果表明RAW-E6吞噬荧光微球的能力显著强于野生型细胞系RAW264.7和对照细胞系RAW-EGFP;菌落计数和激光共聚焦分析表明RAW-E6细胞系吞噬E.coli的能力也显著强于RAW264.7和RAW-EGFP。【结论】通过胞内表达发现结核分枝杆菌分泌蛋白ESAT-6能够增强巨噬细胞的吞噬功能,这将为深入理解结核分枝杆菌的致病机制提供新的思路。     

The lipid kinase PI4KIIIβ preserves lysosomal identity

Lipid modifications are essential in cellular sorting and trafficking inside cells. The role of phosphoinositides in trafficking between Golgi and endocytic/lysosomal compartments has been extensively explored and the kinases responsible for these lipid changes have been identified. In contrast, the mechanisms that mediate exit and recycling from lysosomes (Lys), considered for a long time as terminal compartments, are less understood. In this work, we identify a dynamic association of the lipid kinase PI4KIIIβ with Lys and unveil its regulatory function in lysosomal export and retrieval. We have found that absence of PI4KIIIβ leads to abnormal formation of tubular structures from the lysosomal surface and loss of lysosomal constituents through these tubules. We demonstrate that the kinase activity of PI4KIIIβ is necessary to prevent this unwanted lysosomal efflux under normal conditions, and to facilitate proper sorting when recycling of lysosomal material is needed, such as in the physiological context of lysosomal reformation after prolonged starvation.