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。     

The thymus as a target for mycobacterial infections

Mycobacterial infections are among the major health threats worldwide. Ability to fight these infections depends on the host's immune response, particularly on macrophages and T lymphocytes produced by the thymus. Using the mouse as a model, and two different routes of infection (aerogenic or intravenous), we show that the thymus is consistently colonized by Mycobacterium tuberculosis, Mycobacterium avium or Mycobacterium bovis BCG. When compared to organs such as the liver and spleen, the bacterial load reaches a plateau at later time-points after infection. Moreover, in contrast with organs such as the spleen and the lung no granuloma were found in the thymus of mice infected with M. tuberculosis or M. avium. Since T cell differentiation depends, to a large extent, on the antigens encountered within the thymus, infection of this organ might alter the host's immune response to infection. Therefore, from now on, the thymus should be considered in studies addressing the immune response to mycobacterial infection.     

巨噬细胞凋亡对结核分枝杆菌感染的机制研究进展

结核病是一种严重危害人类健康的慢性传染性疾病,主要由结核分枝杆菌感染导致,结核分枝杆菌进入人体后,与免疫防御的第一道屏障—巨噬细胞发生反应,部分菌株在细胞内长期生存、繁殖,是导致结核病转归的决定性因素。感染早期,结核分枝杆菌的繁殖受到巨噬细胞凋亡的抑制,随着高效价、高毒力菌株繁殖速度的增加,抗巨噬细胞凋亡作用不断增强,使自身繁殖得到有效保护,为菌株的生长提供了充足、适宜的胞内环境。因此,调控结核分枝杆菌对巨噬细胞凋亡进程的抑制作用,是预防和治疗结核病的关键。     

Effects of Mycobacterium tuberculosis ESAT-6/CFP-10 fusion protein on the autophagy function of mouse macrophages

Autophagy plays specific roles in host innate and adaptive immune responses to numerous intracellular pathogens, including Mycobacterium tuberculosis. The ESAT-6 and CFP-10 proteins are secreted by M. tuberculosis and play important roles in pathogenesis. We hypothesized that these two proteins may affect the autophagy function of host macrophages during infection with M. tuberculosis, thereby shaping the immune reaction toward the pathogen. Interestingly, we found that rapamycin-induced autophagy of macrophages infected with M. tuberculosis H37Rv enhanced localization of mycobacteria with autophagosomes and lysosomes. Ectopic expression of the ESAT-6/CFP-10 fusion in macrophages dramatically inhibited autophagosome formation, and M. tuberculosis survival inside infected macrophages was significantly affected as well. Further, M. tuberculosis viability was increased by the fusion protein. Expression levels of autophagy-related genes (ATG), especially atg8, also decreased (p<0.05). These results suggested that ESAT-6 and CFP-10 proteins play significant roles in autophagy formation in M. tuberculosis infection and that autophagosome formation is regulated through the expression of ATG.     

Relative levels of M-CSF and GM-CSF influence the specific generation of macrophage populations during infection with Mycobacterium tuberculosis

Members of the CSF cytokine family play important roles in macrophage recruitment and activation. However, the role of M-CSF in pulmonary infection with Mycobacterium tuberculosis is not clear. In this study, we show the lungs of mice infected with M. tuberculosis displayed a progressive decrease in M-CSF in contrast to increasing levels of GM-CSF. Restoring pulmonary M-CSF levels during infection resulted in a significant decrease in the presence of foamy macrophages and increased expression of CCR7 and MHC class II, specifically on alveolar macrophages. In response to M-CSF, alveolar macrophages also increased their T cell-stimulating capacity and expression of DEC-205. These studies show that the levels of expression of M-CSF and GM-CSF participate in the progression of macrophages into foamy cells and that these cytokines are important factors in the differentiation and regulation of expression of dendritic cell-associated markers on alveolar macrophages. In addition, these studies demonstrate that M-CSF may have a role in the adaptive immune response to infection with M. tuberculosis.     

Production of phthiocerol dimycocerosates protects Mycobacterium tuberculosis from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection

The growth of Mycobacterium tuberculosis mutants unable to synthesize phthiocerol dimycocerosates (DIMs) was recently shown to be impaired in mouse lungs. However, the precise role of these molecules in the course of infection remained to be determined. Here, we provide evidence that the attenuation of a DIM-deficient strain takes place during the acute phase of infection in both lungs and spleen of mice, and that this attenuation results in part from the increased sensitivity of the mutant to the cidal activity of reactive nitrogen intermediates released by activated macrophages. We also show that the DIM-deficient mutant, the growth and survival of which were not impaired within resting macrophages and dendritic cells, induced these cells to secrete more tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 than the wild-type strain. Although purified DIM molecules by themselves had no effect on the activation of macrophages and dendritic cells in vitro, we found that the proper localization of DIMs in the cell envelope of M. tuberculosis is critical to their biological effects. Thus, our findings suggest that DIM production contributes to the initial growth of M. tuberculosis by protecting it from the nitric oxide-dependent killing of macrophages and modulating the early immune response to infection.     

Mycobacterium tuberculosis subverts innate immunity to evade specific effectors

The macrophage is the niche of the intracellular pathogen Mycobacterium tuberculosis. Induction of macrophage apoptosis by CD4(+) or CD8(+) T cells is accompanied by reduced bacterial counts, potentially defining a host defense mechanism. We have already established that M. tuberculosis-infected primary human macrophages have a reduced susceptibility to Fas ligand (FasL)-induced apoptosis. To study the mechanisms by which M. tuberculosis prevents apoptotic signaling, we have generated a cell culture system based on PMA- and IFN-gamma-differentiated THP-1 cells recapitulating the properties of primary macrophages. In these cells, nucleotide-binding oligomerization domain 2 or TLR2 agonists and mycobacterial infection protected macrophages from apoptosis and resulted in NF-kappaB nuclear translocation associated with up-regulation of the antiapoptotic cellular FLIP. Transduction of a receptor-interacting protein-2 dominant-negative construct showed that nucleotide-binding oligomerization domain 2 is not involved in protection in the mycobacterial infection system. In contrast, both a dominant-negative construct of the MyD88 adaptor and an NF-kappaB inhibitor abrogated the protection against FasL-mediated apoptosis, showing the implication of TLR2-mediated activation of NF-kappaB in apoptosis protection in infected macrophages. The apoptosis resistance of infected macrophages might be considered as an immune escape mechanism, whereby M. tuberculosis subverts innate immunity signaling to protect its host cell against FasL(+)-specific cytotoxic lymphocytes.     

Mycobacterium tuberculosis triggers host type I IFN signaling to regulate IL-1β production in human macrophages

Mycobacterium tuberculosis is a virulent intracellular pathogen that survives in macrophages even in the presence of an intact adaptive immune response. Type I IFNs have been shown to exacerbate tuberculosis in mice and to be associated with disease progression in infected humans. Nevertheless, the mechanisms by which type I IFNs regulate the host response to M. tuberculosis infection are poorly understood. In this study, we show that M. tuberculosis induces an IFN-related gene expression signature in infected primary human macrophages, which is dependent on host type I IFN signaling as well as the mycobacterial virulence factor, region of difference-1. We further demonstrate that type I IFNs selectively limit the production of IL-1β, a critical mediator of immunity to M. tuberculosis. This regulation occurs at the level of IL1B mRNA expression, rather than caspase-1 activation or autocrine IL-1 amplification and appears to be preferentially used by virulent mycobacteria since avirulent M. bovis bacillus Calmette-Guérin (BCG) fails to trigger significant expression of type I IFNs or release of mature IL-1β protein. The latter property is associated with decreased caspase-1-dependent IL-1β maturation in the BCG-infected macrophages. Interestingly, human monocytes in contrast to macrophages produce comparable levels of IL-1β in response to either M. tuberculosis or BCG. Taken together, these findings demonstrate that virulent and avirulent mycobacteria employ distinct pathways for regulating IL-1β production in human macrophages and reveal that in the case of M. tuberculosis infection the induction of type I IFNs is a major mechanism used for this purpose.     

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.     

Mycobacterial mutants with defective control of phagosomal acidification

The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.     

ATP stimulates human macrophages to kill intracellular virulent Mycobacterium tuberculosis via calcium-dependent phagosome-lysosome fusion.

Advances in therapy for tuberculosis will require greater understanding of the molecular mechanisms of pathogenesis and the human immune response in this disease. Exposure of Mycobacterium tuberculosis-infected human macrophages to extracellular ATP (ATP(e)) results in bacterial killing, but the molecular mechanisms remain incompletely characterized. In this study, we demonstrate that ATP(e)-induced bactericidal activity toward virulent M. tuberculosis requires an increase in cytosolic Ca(2+) in infected macrophages. Based on our previous work with primary infection of human macrophages, we hypothesized that the Ca(2+) dependence of ATP-induced killing of intracellular M. tuberculosis was linked to promotion of phagosome-lysosome fusion. Using confocal laser-scanning microscopy, we demonstrate that ATP(e) induces fusion of the M. tuberculosis-containing phagosome with lysosomes, defined by accumulation of three lysosomal proteins and an acidophilic dye. Stimulation of phagosome-lysosome fusion by ATP(e) exhibited distinct requirements for both Ca(2+) and phospholipase D and was highly correlated with killing of intracellular bacilli. Thus, key signal transduction pathways are conserved between two distinct models of human macrophage antituberculous activity: primary infection of naive macrophages and physiologic stimulation of macrophages stably infected with M. tuberculosis.     

Cyclic AMP signalling in mycobacteria: redirecting the conversation with a common currency

cAMP is an ancient second messenger, and is used by many organisms to regulate a wide range of cellular functions. Mycobacterium tuberculosis complex bacteria are exceptional in that they have genes for at least 15 biochemically distinct adenylyl cyclases, the enzymes that generate cAMP. cAMP-associated gene regulation within tubercle bacilli is required for their virulence, and secretion of cAMP produced by M. tuberculosis bacteria into host macrophages disrupts the host's immune response to infection. In this review, we discuss recent advances in our understanding of the means by which cAMP levels are controlled within mycobacteria, the importance of cAMP to M. tuberculosis during host infection, and the role of cAMP in mycobacterial gene regulation. Understanding the myriad aspects of cAMP signalling in tubercle bacilli will establish new paradigms for cAMP signalling, and may contribute to new approaches for prevention and/or treatment of tuberculosis disease.     

Mycobacterium tuberculosis induces differential cytokine production from dendritic cells and macrophages with divergent effects on naive T cell polarization

Th1-mediated cellular responses are important for protection in tuberculosis. However, the mechanisms and APC types responsible for initiating Th1 responses are not well understood. These studies show that macrophages and dendritic cells, albeit both being APC, respond differently following Mycobacterium tuberculosis infection and thereby have different consequences for the development of naive T cells. We report that M. tuberculosis-infected dendritic cells bias the polarization of OVA peptide-specific naive transgenic T cells to the Th1 phenotype, and, in contrast, in the presence of infected macrophages naive T cells do not develop a Th1 phenotype. Comparison of the cytokine profile expressed by the infected dendritic cells and macrophages revealed several differences, the most striking being that infected macrophages did not express the Th1-promoting cytokine IL-12. These studies also show that IL-10 is responsible for the failure of IL-12 production by M. tuberculosis-infected macrophages, and that the effects of IL-10 can be overcome by IFN-gamma priming. We speculate that the observed difference in response of the two APC types to M. tuberculosis infection may be a reflection of their respective roles in immune initiation and granuloma regulation.     

Silencing suppressor of cytokine signaling-1 (SOCS1) in macrophages improves Mycobacterium tuberculosis control in an interferon-gamma (IFN-gamma)-dependent manner

Protection against infection with Mycobacterium tuberculosis demands IFN-γ. SOCS1 has been shown to inhibit responses to IFN-γ and might thereby play a central role in the outcome of infection. We found that M. tuberculosis is a highly efficient stimulator of SOCS1 expression in murine and human macrophages and in tissues from infected mice. Surprisingly, SOCS1 reduced responses to IL-12, resulting in an impaired IFN-γ secretion by macrophages that in turn accounted for a deteriorated intracellular mycobacterial control. Despite SOCS1 expression, mycobacteria-infected macrophages responded to exogenously added IFN-γ. SOCS1 attenuated the expression of the majority of genes modulated by M. tuberculosis infection of macrophages. Using a conditional knockdown strategy in mice, we found that SOCS1 expression by macrophages hampered M. tuberculosis clearance early after infection in vivo in an IFN-γ-dependent manner. On the other hand, at later time points, SOCS1 expression by non-macrophage cells protected the host from infection-induced detrimental inflammation.     

CCR5-deficient mice control Mycobacterium tuberculosis infection despite increased pulmonary lymphocytic infiltration

The control of Mycobacterium tuberculosis infection is dependent on the development of an adaptive immune response, which is mediated by granulomas. The granuloma is a dynamic structure that forms in the lung and consists primarily of macrophages and lymphocytes. For this structure to be effective in containment of the bacillus, it must develop in an organized and timely manner. The formation of the granuloma is dependent on recruitment of activated cells through adhesion molecules and chemokines. M. tuberculosis infection causes an increase in the expression of beta-chemokines CCL3, CCL4, and CCL5, and their receptor CCR5, in the lungs. In this study, we demonstrate that CCR5-transgenic knockout mice were capable of recruiting immune cells to the lung to form granulomas. CCR5(-/-) mice successfully induced a Th1 response and controlled infection. Unexpectedly, M. tuberculosis infection in these mice resulted in greater numbers of lymphocytes migrating to the lung and higher levels of many inflammatory cytokines, compared with wild-type mice, without apparent long-term detrimental effects. In the absence of CCR5, there were more dendritic cells in the lung-draining lymph nodes and more primed T lymphocytes in these mice. Bacterial numbers in the lymph nodes were also higher in CCR5(-/-) mice. Therefore, CCR5 may play a role in the migration of dendritic cells to and from the lymph nodes during M. tuberculosis infection.     

TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection

Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF(-/-)) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b(+) macrophages and CD4(+) T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF(-/-) mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF(-/-) mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF(-/-) mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.     

Aging and immunity to tuberculosis: increased susceptibility of old mice reflects a decreased capacity to generate mediator T lymphocytes

This study employed an experimental mouse model of Mycobacterium tuberculosis infection to investigate the effects of aging on T cell-mediated protective cellular immunity. It was found that although mice of 3 to 18 mo of age were fully resistant to a standard immunizing dose of Mycobacterium tuberculosis, progressive mortality was observed in old (24 to 28 mo) mice. Death of these older animals was associated with an inability to contain or to eliminate the mycobacterial infection in the spleen and liver, and with an inability to prevent the progressive growth of the infection in the lungs. It was then revealed by the use of reciprocal passive cell transfer experiments that the age-related susceptibility of old mice reflected an inability to generate mediator protective T lymphocytes in response to the infection. In contrast, no evidence was obtained to indicate any defect at the effector cell (macrophage) level, as evidenced primarily by the finding that immune T cells from young mice conferred equivalent levels of immunity upon both old and young recipients. The results suggest therefore that T cell-mediated immunity undergoes an age-related decline in terms of its ability to respond to infection with Mycobacterium tuberculosis.     

Mycobacterium bovis bacillus Calmette-Guérin induces TLR2-mediated formation of lipid bodies: intracellular domains for eicosanoid synthesis in vivo

Differentiation of macrophages into foamy (lipid-laden) macrophages is a common pathological observation in tuberculous granulomas both in experimental settings as well as in clinical conditions; however, the mechanisms that regulate intracellular lipid accumulation in the course of mycobacterial infection and their significance to pathophysiology of tuberculosis are not well understood. In this study, we investigated the mechanisms of formation and function of lipid-laden macrophages in a murine model of tuberculosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG), but not Mycobacterium smegmatis, induced a dose- and time-dependent increase in lipid body-inducible nonmembrane-bound cytoplasmic lipid domain size and numbers. Lipid body formation was drastically inhibited in TLR2-, but not in TLR4-deficient mice, indicating a role for TLR2 in BCG recognition and signaling to form lipid bodies. Increase in lipid bodies during infection correlated with increased generation of PGE2 and localization of cyclooxygenase-2 within lipid bodies. Moreover, we demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid that lipid bodies were the predominant sites of PGE2 synthesis in activated macrophages. Our findings demonstrated that BCG-induced lipid body formation is TLR2 mediated and these structures function as signaling platforms in inflammatory mediator production, because compartmentalization of substrate and key enzymes within lipid bodies has impact on the capacity of activated leukocytes to generate increased amounts of eicosanoids during experimental infection by BCG.     

CD8+ CTL from lungs of Mycobacterium tuberculosis-infected mice express perforin in vivo and lyse infected macrophages

CD8+ T lymphocytes have been implicated in the protective immune response against human and murine tuberculosis. However, the functional role that this cell subset plays during the resolution of infection remains controversial. In this study, we demonstrate the presence of Mycobacterium tuberculosis-specific CD8+ CTL in the lungs and lung-draining lymph nodes of mice infected with M. tuberculosis via the aerosol or i.v. route. These cells expressed perforin in vivo and specifically recognized and lysed M. tuberculosis-infected macrophages in a perforin-dependent manner after a short period of in vitro restimulation. The efficiency of lysis of infected macrophages was dependent upon the time allowed for interaction between macrophage and M. tuberculosis bacilli. Recognition of infected targets by CD8+ CTL was beta 2-microglobulin and MHC class I dependent and was not CD1d restricted. The presented data indicate that CD8+ T cells contribute to the protective immune response during M. tuberculosis infection by exerting cytotoxic function and lysing infected macrophages.     

Infection biology of a novel alpha-crystallin of Mycobacterium tuberculosis: Acr2

Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the alpha-crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (Deltaacr2) was unimpaired in log phase growth and persisted in IFN-gamma-activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN-gamma-secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.