Mycobacterium tuberculosis diverts alpha interferon-induced monocyte differentiation from dendritic cells into immunoprivileged macrophage-like host cells

Dendritic cells (DCs) are critical for initiating a pathogen-specific T-cell response. During chronic infections the pool of tissue DCs must be renewed by recruitment of both circulating DC progenitors and in loco differentiating monocytes. However, the interaction of monocytes with pathogens could affect their differentiation. Mycobacterium tuberculosis has been shown to variably interfere with the generation and function of antigen-presenting cells (APCs). In this study we found that when alpha interferon (IFN-alpha) is used as an inductor of monocyte differentiation, M. tuberculosis inhibits the generation of DCs, forcing the generation of immunoprivileged macrophage-like cells instead. Cells derived from M. tuberculosis-infected monocyte-derived macrophages (M. tuberculosis-infected MoMphi) retained CD14 without acquiring CD1 molecules and partially expressed B7.2 but did not up-regulate B7.1 and major histocompatibility complex (MHC) class I and II molecules. They synthesized tumor necrosis factor alpha and interleukin-10 (IL-10) but not IL-12. They also showed a reduced ability to induce proliferation and functional polarization of allogeneic T lymphocytes. Thus, in the presence of IFN-alpha, M. tuberculosis may hamper the renewal of potent APCs, such as DCs, generating a safe habitat for intracellular growth. M. tuberculosis-infected MoMphi, in fact, showed reduced expression of both signal 1 (CD1, MHC classes I and II) and signal 2 (B7.1 and B7.2), which are essential for mycobacterium-specific T-lymphocyte priming and/or activation. These data further suggest that M. tuberculosis has the ability to specifically interfere with monocyte differentiation. This ability may represent an effective M. tuberculosis strategy for eluding immune surveillance and persisting in the host.     

Stimulation of antibacterial macrophage activities by B-cell stimulatory factor 2 (interleukin-6).

Mononuclear phagocytes provide the major habitat of intracellular bacteria, including Mycobacterium tuberculosis and Mycobacterium bovis. The capacity of B-cell stimulatory factor 2 (interleukin-6 [IL-6]) to activate tuberculostatic functions was investigated by using murine bone marrow-derived macrophages (BMM phi). BMM phi stimulated with recombinant IL-6 and subsequently infected with M. bovis organisms failed to inhibit mycobacterial growth. In contrast, marked tuberculostasis was induced by IL-6 in BMM phi that were already infected with M. bovis, indicating that IL-6 has a macrophage-activating function.     

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.     

Comparison of in vitro models for the study of Mycobacterium tuberculosis invasion and intracellular replication.

We recently evaluated several tissue culture model systems for the study of invasion and intracellular multiplication of Mycobacterium tuberculosis. These model systems include a human alveolar pneumocyte epithelial cell line, a murine macrophage cell line (J774), and fresh human peripheral blood-derived macrophages. Our data indicated that the initial level of association of M. tuberculosis with human alveolar pneumocyte cells (2%) was less than that observed with fresh human peripheral blood macrophages (9%) or J774 murine macrophages (13%) within 6 h of the addition of the bacteria. M. tuberculosis replicated in association with the pneumocyte cells by more than 55-fold by day 7 postinfection. In contrast, total bacteria] growth in the J774 cells and human macrophages was considerably less, with increases of only fourfold and threefold, respectively, over the same 7-day period. Amikacin, an aminoglycoside antimicrobial agent, was added to inhibit the growth of extracellular bacteria after the initial 6-h infection period. Decreases in viable counts were observed in all three cell cultures within the first 3 days after infection. However, unlike the case with either macrophage culture, intracellular bacterial CFU obtained from the infected pneumocytes increased by fourfold by day 7 after the addition of amikacin. These data indicate that M. tuberculosis infects and multiplies intracellularly in human lung epithelial cells and that these cells may be an alternative in vitro model for the study of intracellular multiplication of M. tuberculosis in the human lung.     

Dendritic cells and Mycobacterium tuberculosis: which is the Trojan horse?

A new scenario has been unraveled recently--the interaction between the human dendritic cell (DC) and Mycobacterium tuberculosis. Whether this encounter represents a defense mechanism by the invaded host, or a smoke screen, masking the presence of an invader is still unknown. The intracellular behavior of M. tuberculosis inside DCs differs compared to macrophages (Mphis), with a failure of replication. The intracellular compartment of the DC, disconnected from the exocytic and endocytic pathways, and characterized by the absence of endoplasmic reticulum and Golgi features, places M. tuberculosis in a hostile environment, where a ready source of nutrients is scarce. The differential behavior inside Mphis and DCs is linked to a different portal of entry. DCs harbor surface lectins receptors, like DC-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN/CD209), a binding site which is absent on Mphis. This receptor interacts exclusively with M. tuberculosis. The ligand is the mannose-capped lipoarabinomanan (LAM), absent from atypical mycobacteria. M. smegmatis, M. chelonae and M. fortuitum, which possess LAM capped with phosphoinositides residues, do not bind to DC-SIGN, demonstrating a role for DC-SIGN as a 'pattern-recognition receptor' with the ability to differentiate between pathogenic and non-pathogenic mycobacteria. Interactions of M. tuberculosis with DC-SIGN have antiinflammatory effects. Whether this property is of benefit to the invader remains to be discovered.     

Colony-stimulating factors activate human macrophages to inhibit intracellular growth of Histoplasma capsulatum yeasts.

Recombinant cytokines and colony-stimulating factors (CSFs) were tested for their abilities to activate human monocytes/macrophages (M phi) to inhibit the intracellular growth of or kill Histoplasma capsulatum yeasts. None of the cytokines or CSFs or combinations of cytokines and CSFs activated M phi fungistatic activity when they were added to M phi monolayers concurrently with yeasts. In contrast, culture of monocytes for 7 days in the presence of interleukin 3, granulocyte-M phi CSF, or M phi CSF stimulated M phi fungistatic (but not fungicidal) activity against H. capsulatum yeasts in a concentration-dependent manner. Optimal activation of M phi by CSFs required 5 days of coculture, and the cultures had to be initiated with freshly isolated peripheral blood monocytes. Culture of monocytes with combinations of CSFs or addition of CSFs during the 24 h of coculture with the yeasts did not further enhance M phi fungistatic activity for H. capsulatum. Addition of gamma interferon or tumor necrosis factor alpha to CSF-activated M phi also did not enhance M phi fungistatic activity. These results suggest that interleukin 3, granulocyte-M phi CSF, and M phi CSF may play a role in the cell-mediated immune response to H. capsulatum by enhancing monocyte/M phi fungistatic activity.     

Macrophage defense mechanisms against intracellular bacteria

Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics.     

Human natural killer cells mediate killing of intracellular Mycobacterium tuberculosis H37Rv via granule-independent mechanisms

Despite the continued importance of tuberculosis as a world-wide threat to public health, little is known about the mechanisms used by human lymphocytes to contain and kill the intracellular pathogen Mycobacterium tuberculosis. We previously described an in vitro model of infection of human monocytes (MN) with virulent M. tuberculosis strain H37Rv in which the ability of peripheral blood lymphocytes to limit intracellular growth of the organism could be measured. In the current study, we determined that lymphocyte-mediated killing of intracellular M. tuberculosis occurs within the first 24 h of coculture with infected MN. Natural killer (NK) cells isolated from both purified protein derivative (PPD)-positive and PPD-negative subjects were capable of mediating this early killing of intracellular H37Rv. NK cell-mediated killing of intracellular M. tuberculosis was not associated with the production of gamma interferon. Transferred supernatants of cocultured NK cells and M. tuberculosis-infected MN could not mediate the killing of intracellular M. tuberculosis, and Transwell studies indicated that direct cell-to-cell contact was required for NK cells to mediate the killing of the organism. Killing was not dependent upon exocytosis of NK cell cytotoxic granules. NK cells induced apoptosis of mycobacterium-infected MN, but neither killing of intracellular M. tuberculosis by NK cells nor NK cell-induced apoptosis of infected MN was inhibited by blocking the interaction of FasL and Fas. Thus, human NK cells may mediate killing of intracellular M. tuberculosis via alternative apoptotic pathways.     

Differences in the growth of paired Ugandan isolates of Mycobacterium tuberculosis within human mononuclear phagocytes correlate with epidemiological evidence of strain virulence

Previous studies have suggested that isolates of Mycobacterium tuberculosis responsible for tuberculosis outbreaks grow more rapidly within human mononuclear phagocytes than do other isolates. Clinical scenarios suggesting virulence of specific M. tuberculosis isolates are readily identified. Determination of appropriate "control" isolates for these studies is more problematic, but equally important for validating these assays and, ultimately, for identifying biologic differences between M. tuberculosis strains that contribute to virulence. We utilized the database from a study of Ugandan tuberculosis patients and their household (HH) contacts to identify M. tuberculosis isolates transmitted within HH and nontransmitted control isolates. Isolate pairs were evaluated from matched HH in each of three clinical scenarios: (i) coprevalent disease and no disease, (ii) incident disease and no disease, and (iii) M. tuberculosis infection (purified protein derivative [PPD] positive) and no infection (PPD negative). Intracellular growth of paired organisms was determined in a blinded fashion using two models of intracellular infection in which we have previously demonstrated correlation between intracellular growth and strain virulence, primary human monocytes (MN) and THP-1 human macrophage-like cells. In both models, transmitted isolates from coprevalent disease HH displayed more rapid growth than nontransmitted control isolates. In the THP-1 model, this was also true of transmitted isolates from HH with incident disease and their controls. Differences in production of tumor necrosis factor alpha and interleukin-10 by matched isolates showed correlation with growth patterns in the THP-1 cells but not in MN. Paired isolates characterized in this manner may be of particular interest for further investigations of the virulence of M. tuberculosis.     

The regulation of macrophage activity in congenitally athymic mice

It has been suggested that non-T cell-mediated cellular immune mechanisms might be elevated in nude mice, which could contribute to their partial resistance to intracellular infectious agents and to the development of spontaneous tumors. In this study we have examined macrophages (M phi) from athymic, euthymic and athymic T cell-reconstituted mice in terms of their phagocytic capacity, microbicidal and tumoricidal activity, and the production of hydrogen peroxidase and superoxide anions. These studies have demonstrated the presence of activated M phi in nude mice and suggest that this activation is associated with the absence of T cell-mediated suppression. We have also compared M phi activation levels in germ-free nude mice which have received a defined intestinal bacterial flora. We have found M phi activity to be significantly elevated in microbiologically defined nude mice when compared to germ-free nude mice, indicating that a resistant gut flora is capable of nonspecifically stimulating nude mouse M phi.     

The glycan-rich outer layer of the cell wall of Mycobacterium tuberculosis acts as an antiphagocytic capsule limiting the association of the bacterium with macrophages

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that infects macrophages and other host cells. We show that sonication of M. tuberculosis results in the removal of material from the surface capsule-like layer of the bacteria, resulting in an enhanced propensity of the bacteria to bind to macrophages. This effect is observed with disparate murine and human macrophage populations though, interestingly, not with freshly explanted alveolar macrophages. Enhanced binding to macrophages following sonication is significantly greater within members of the M. tuberculosis family (pathogens) than within the Mycobacterium avium complex (opportunistic pathogens) or for Mycobacterium smegmatis (saprophyte). Sonication does not affect the viability or the surface hydrophobicity of M. tuberculosis but does result in changes in surface charge and in the binding of mannose-specific lectins to the bacterial surface. The increased binding of sonicated M. tuberculosis was not mediated through complement receptor 3. These results provide evidence that the surface capsule on members of the M. tuberculosis family may be an important virulence factor involved in the survival of M. tuberculosis in the mammalian host. They also question the view that M. tuberculosis is readily ingested by any macrophage it encounters and support the contention that M. tuberculosis, like many other microbial pathogens, has an antiphagocytic capsule that limits and controls the interaction of the bacterium with macrophages.     

Implication of phagosome-lysosome fusion in restriction of Mycobacterium avium growth in bone marrow macrophages from genetically resistant mice.

The ability of the host to resist infection to a variety of intracellular pathogens, including mycobacteria, is strongly dependent upon the expression of the Bcg gene. Mouse strains which express the resistance phenotype (Bcgr) restrict bacterial growth, whereas susceptible strains (Bcgs) allow bacterial growth. Expression of the Bcg allele is known to influence the priming of host macrophages (M phi s) for bactericidal function. In the present work, bone marrow-derived M phi s from congenic BALB/c (Bcgs) and C.D2 (BALB/c.Bcgr) mice were infected with the virulent strain Mycobacterium avium TMC 724 to define the mechanism involved in growth restriction of M. avium. By combining CFU measurements and ultrastructural analyses, we show that growth of this bacterium is restricted in marrow M phi s from resistant mice. Using acid phosphatase as a lysosomal marker, we provide evidence that the hydrolytic activity of M phi s, as measured by the capacity of lysosomes to fuse with and transfer active hydrolytic enzymes to phagosomes in which M. avium resides, is an expression of the Bcg gene and that this phenomenon is a key antibacterial activity responsible for growth restriction of M. avium: (i) the percentage of phagosome-lysosome fusions was twice as high in Bcgr M phi s as in Bcgs M phi s, and (ii) the percentage of intact viable bacteria residing in acid phosphatase-negative phagosomes was twice as low in Bcgr M phi s as in the Bcgs counterparts. These differences are not due to a lower activity of the enzyme in Bcgr M phi s. The mechanism by which the Bcg gene exerts control over the phagolysosomal fusion is discussed.     

Gallium disrupts iron metabolism of mycobacteria residing within human macrophages

Mycobacterium tuberculosis and M. avium complex (MAC) enter and multiply within monocytes and macrophages in phagosomes. In vitro growth studies using standard culture media indicate that siderophore-mediated iron (Fe) acquisition plays a critical role in the growth and metabolism of both M. tuberculosis and MAC. However, the applicability of such studies to conditions within the macrophage phagosome is unclear, due in part to the absence of experimental means to inhibit such a process. Based on the ability of gallium (Ga(3+)) to concentrate within mononuclear phagocytes and on evidence that Ga disrupts cellular Fe-dependent metabolic pathways by substituting for Fe(3+) and failing to undergo redox cycling, we hypothesized that Ga could disrupt Fe acquisition and Fe-dependent metabolic pathways of mycobacteria. We find that Ga(NO(3))(3) and Ga-transferrin produce an Fe-reversible concentration-dependent growth inhibition of M. tuberculosis strains and MAC grown extracellularly and within human macrophages. Ga is bactericidal for M. tuberculosis growing extracellularly and within macrophages. Finally, we provide evidence that exogenously added Fe is acquired by intraphagosomal M. tuberculosis and that Ga inhibits this Fe acquisition. Thus, Ga(NO(3))(3) disruption of mycobacterial Fe metabolism may serve as an experimental means to study the mechanism of Fe acquisition by intracellular mycobacteria and the role of Fe in intracellular survival. Furthermore, given the inability of biological systems to discriminate between Ga and Fe, this approach could have broad applicability to the study of Fe metabolism of other intracellular pathogens.     

Beta-chemokines are induced by Mycobacterium tuberculosis and inhibit its growth

Chemokines (CK) are potent leukocyte activators and chemoattractants and aid in granuloma formation, functions critical for the immune response to Mycobacterium tuberculosis. We hypothesized that infection of alveolar macrophages (AM) with different strains of M. tuberculosis elicits distinct profiles of CK, which could be altered by human immunodeficiency virus (HIV) infection. RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and MIP-1 beta were the major beta-CK produced in response to M. tuberculosis infection. Virulent M. tuberculosis (H37Rv) induced significantly less MIP-1 alpha than did the avirulent strain (H37Ra), while MIP-1 beta and RANTES production was comparable for both strains. MIP-1 alpha and MIP-1 beta were induced by the membrane, but not cytosolic, fraction of M. tuberculosis. M. tuberculosis-induced CK secretion was partly dependent on tumor necrosis factor alpha (TNF-alpha). AM from HIV-infected individuals produced less TNF-alpha and MIP-1 beta than did normal AM in response to either M. tuberculosis strain. We tested the functional significance of decreased beta-CK secretion by examining the ability of beta-CK to suppress intracellular growth of M. tuberculosis. MIP-1 beta and RANTES suppressed intracellular growth of M. tuberculosis two- to threefold, a novel finding. Thus, beta-CK contribute to the innate immune response to M. tuberculosis infection, and their diminution may promote the intracellular survival of M. tuberculosis.     

Nocardia farcinica Activates Human Dendritic Cells and Induces Secretion of Interleukin-23 (IL-23) Rather than IL-12p70

Studying the interaction of dendritic cells (DCs) with bacteria controlled by T-cell-mediated immune responses may reveal novel adjuvants for the induction of cellular immunity. Murine studies and the observation that nocardias infect predominantly immunosuppressed patients have suggested that these bacteria may possess an adjuvant potential. Moreover, adjuvants on the basis of the nocardial cell wall have been applied in clinical studies. Since the handling of adjuvants by DCs may determine the type of immune responses induced by a vaccine, the present study aimed at investigating the interaction of immature human monocyte-derived DCs with live or inactivated Nocardia farcinica in vitro and determining the cellular phenotypic changes as well as alterations in characteristic functions, such as phagocytosis, induction of T-cell proliferation, and cytokine secretion. Human DCs ingested N. farcinica and eradicated the bacterium intracellularly. DCs exposed to inactivated N. farcinica were activated, i.e., they developed a mature phenotype, downregulated their phagocytic capacity, and stimulated allogeneic T cells in mixed leukocyte reactions. Soluble factors were not involved in this process. To elucidate the potential adjuvant effect of N. farcinica on the induction of T-cell-mediated immune responses, we characterized the cytokines produced by nocardia-exposed DCs and detected substantial amounts of tumor necrosis factor alpha (TNF-α) and interleukin-12 p40 (IL-12p40). However, nocardia-treated DCs secreted only small amounts of IL-12p70, which were significantly smaller than the amounts of IL-23. Thus, N. farcinica activates DCs, but adjuvants based on this bacterium may have only a limited capacity to induce Th1 immune responses.     

S-layer-mediated association of Aeromonas salmonicida with murine macrophages.

The interaction of Aeromonas salmonicida with the murine macrophage (M phi) cell line P388D1 was used as a convenient model to study the involvement of the bacterial crystalline surface array (or A-layer) in the association with M phi s. A-layer-positive (A+) cells readily associated with M phi s in phosphate-buffered saline, whereas A- mutants were unable to do so, even when the bacterium-M phi interaction was forced by centrifugation. M phi s selectively interacted with A+ cells when challenged with mixtures of A+ and excess A- cells. Electron microscopy indicated that in phosphate-buffered saline only A+ bacteria were readily internalized, although by a nonconventional mechanism, suggesting that efficient phagocytosis in the absence of opsonins was A-layer mediated. Latex beads coated with a partially assembled A-layer were more efficiently taken up than uncoated or A-protein-coated beads, indicating that an organized A-layer was essential for M phi uptake. The reduced ability of M phi s plated on a substratum coated with the A-layer to bind A+ bacteria also suggested that association was both A-layer and receptor mediated. In the presence of tissue culture medium, competent M phi s interacted efficiently with A- bacteria and internalized them through conventional phagocytosis. A+ cells were markedly cytotoxic to M phi s, whereas the A-protein or A-layer was not. A- cells were cytotoxic to a lesser extent, suggesting that cytotoxicity was targeted.     

The Mycobacterium tuberculosis phagosome in human macrophages is isolated from the host cell cytoplasm

Knowledge of whether Mycobacterium tuberculosis resides within a relatively impermeable membrane-bound vacuole or is free within the cytoplasm within its host cell is central to an understanding of the immunobiology of this intracellular parasite but is a matter of controversy. To explore this issue, we assessed the accessibility of medium-size protein molecules (Fab fragments of 50,000 Da) to M. tuberculosis within human macrophages. We infected the macrophages with wild-type or green fluorescent protein-expressing M. tuberculosis, microinjected Fab fragments directed against a major surface antigen of M. tuberculosis into the host cell, and assayed the accessibility of the bacteria to the Fab fragments by both immunofluorescence microscopy and immunogold electron microscopy. Whereas microinjected intact immunoglobulin G molecules against cytoplasmic early endosomal antigen 1 readily stained this antigen, microinjected Fab fragments against M. tuberculosis did not stain the bacterium within its phagosome. In contrast, microinjected Fab fragments against Listeria monocytogenes, an intracellular bacterium known to permeabilize its phagosomal membrane, strongly stained this bacterium. Our study shows that M. tuberculosis resides in an isolated phagosome that is relatively impermeable to cytoplasmic constituents.     

Migration of Salmonella typhimurium --harboring bone marrow--derived dendritic cells towards the chemokines CCL19 and CCL21

Macrophages are considered as main cellular target encountered by the facultative intracellular bacterium Salmonella typhimurium. However, in orally infected mice these pathogens are first internalized by dendritic cells (DCs) that are located in the subepithelial dome of Peyer's patches. Moreover, DCs can penetrate the intestinal epithelium to sample bacteria. Here, we examined the interaction of Salmonella with bone marrow-derived DCs (BM-DCs). In order to study the role of DCs as vehicles for the dissemination of Salmonella, an in vitro model was established. In this model, Salmonella -activated BM-DCs enhanced surface expression of MHC class II and co-stimulatory molecules. We found that, upon maturation, BM-DCs upregulated chemokine receptor 7 (CCR7) mRNA and surface molecule expression. Salmonella -exposed DCs as well as mature DCs, but not immature DCs, were recruited towards the CC chemokines CCL19 and CCL21, two ligands of CCR7. The maturation process of DCs did neither require bacterial internalization nor viability. About one third of the migrated BM-DCs harbored intracellular bacteria, whereas the remaining two third did not contain bacteria. Salmonella, but not an apathogenic E. coli laboratory strain was capable to survive within BM-DCs. Taken together, our data implicate that DCs are first activated and subsequently utilized as carriers by Salmonella.     

Effects of DNA- and Mycobacterium bovis BCG-based delivery of the Flt3 ligand on protective immunity to Mycobacterium tuberculosis

The control of intracellular pathogens such as Mycobacterium tuberculosis is dependent on the activation and maintenance of pathogen-reactive T cells. Dendritic cells (DCs) are the major antigen-presenting cells initiating antimycobacterial T-cell responses in vivo. To investigate if immunization strategies that aim to optimize DC function can improve protective immunity against virulent mycobacterial infection, we exploited the ability of the hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (Flt3L) to expand the number of DCs in vivo. A DNA fusion of the genes encoding murine Flt3L and M. tuberculosis antigen 85B stimulated enhanced gamma interferon (IFN-gamma) release by T cells and provided better protection against virulent M. tuberculosis than DNA encoding the single components. Vaccination of mice with a recombinant Mycobacterium bovis BCG strain secreting Flt3L (BCG:Flt3L) led to early expansion of DCs compared to immunization with BCG alone, and this effect was associated with increased stimulation of BCG-reactive IFN-gamma-secreting T cells. BCG and BCG:Flt3L provided similar protective efficacies against low-dose aerosol M. tuberculosis; however, immunization of immunodeficient mice revealed that BCG:Flt3L was markedly less virulent than conventional BCG. These results demonstrate the potential of in vivo targeting of DCs to improve antimycobacterial vaccine efficacy.     

Visceral adipose tissue specific persistence of Mycobacterium tuberculosis may be reason for the metabolic syndrome

Mycobacterium tuberculosis (Mtb) is highly successful intracellular pathogen. Infection is maintained in spite of acquired immunity and resists eradication by antimicrobials. Following bacillaemia, small numbers of bacteria are disseminated to the extrapulmonary organs most likely including visceral adipose tissue by a mechanism that may involve the migration of M. tuberculosis within dendritic cells. In this lipid rich environment, Mtb can metabolize the fatty acids in a glyoxylate cycle dependent manner, and a state of chronic persistence may ensue. The persistent bacilli primarily use fatty acids as their carbon source. Expression of isocitrate lyase (ICL), gating enzyme of glyoxylate cycle, is upregulated during infection. ICL is important for survival during the persistent phase of infection. Expression of adipokines, particularly monocyte chemoattractant protein-1 (MCP-1), which is a potent proinflammatory cytokine, may be increased. MCP-1 contributes both to the recruitment of macrophages to adipose tissue and to the development of insulin resistance in humans. In addition, prolonged low level immune stimulation induces local adipolipogenesis, increasing visceral fat. Increased delivery of free fatty acid to the liver may stimulate the glyoxylate cycle-induced gluconeogenesis, raising hepatic glucose output. Hence, inhibition of the triggering enzyme ICL, which initiates all the pathologies related to persistent Mtb infection, may block the growth of the bacteria and may resolve the systemic metabolic complications.