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Who puts the tubercle in tuberculosis?

David G. Russell

Abstract | Tuberculosis (TB), an illness that mainly affects the respiratory system, is one of the worlds most pernicious diseases. TB currently infects one-third of the worlds population and kills approximately 1.7 million people each year. Most infected individuals fail to progress to full-blown disease because the TB bacilli are walled off by the immune system inside a tissue nodule known as a granuloma. The granulomas primary function is one of containment and it prevents the dissemination of the mycobacteria. But what is the role of the TB bacillus in the progression of the granuloma? This Review explores how Mycobacterium tuberculosis influences granuloma formation and maintenance, and ensures the spread of the disease.
Founder strain
The ancestral species or strain that underwent divergent evolution to produce several new species or strains.

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA. e-mail: dgr8@cornell.edu doi:10.1038/nrmicro1538 Published online 11 December 2006

The bacterium Mycobacterium tuberculosis is the causative agent of one of those human conditions that, despite the developments of modern medicine, is reluctant to relinquish its grip on mankind. The incidence of tuberculosis (TB) correlates strongly with reduced socio-economic status, thereby adding to the burden of the global poor1,2. Estimates of host penetrance based on skin-test conversion indicate that up to one-third of the worlds population are infected with this bacterium. Over their lifetime it is projected that an infected individual has a 510% risk of developing TB3, although this scenario shows extraordinary regional variation that is impacted to a tragic extent by HIV infection4. Given that such a relatively low number of individuals develop disease and therefore the capacity to transmit the infection, it follows that the transmission process must be supremely efficient to sustain such levels of infection in its host population. The progression of the disease is determined at the level of the infection site itself. Once the bacterium has gained entry into a macrophage and triggered that host cell to invade the tissue of the lung, the host responds by remodelling the site of infection into a cellular mass, the tubercle or granuloma that has given the disease its name. The human tuberculosis granuloma is a beautiful and fascinating structure in which all the acts of the disease are written and performed, as documented in BOX 1. The bulk of the literature on the tuberculosis granuloma focuses on the host component and ignores any influence that the bacterium might have on its progression. However, given that transmission is key to the success of the bacterium, it would be short-sighted to imagine that M. tuberculosis has not evolved specific strategies and effectors to maximize progression to a productive infection. This Review attempts to redress the balance in favour of M. tuberculosis.

What is now ancient history To appreciate the biological significance of the granuloma it is important to consider tuberculosis in the context of human evolution rather than focusing exclusively on the current manifestations of the disease. How has TB impacted on mankind through pre-history? Until recently, it was thought that M. tuberculosis evolved from Mycobacterium bovis and was acquired by humans during the development of agriculture in the Fertile Crescent around 9,000 years ago5, a hypothesis popularized in the book Guns, Germs and Steel by Jared Diamond6. In this scenario, TB would have been introduced into a subpopulation of mankind after their emergence from Africa and one would have predicted, unless other infectious diseases provided comparable selective pressures on fitness and reproduction, that the current human populations would exhibit extreme traits of susceptibility However, this is not the case with the exception of a few extremely rare mutations in central immune system function7, the population genetics of susceptibility to TB reveal relatively modest associations of individual genotypes with disease progression713. More recent studies into the evolution of M. tuberculosis have indicated that M. tuberculosis is actually more closely related to a common founder strain than it is to M. bovis. This implies that either humans infected cattle with M. tuberculosis, resulting in the divergence of M. bovis, or the two strains evolved in parallel from a founder strain that infected both humans and cattle1416. The ancestor of M. tuberculosis most likely arose from the M. tuberculosis-like species of the M. tuberculosis complex that are found today in central Africa1417. This might indicate that humans were exposed to tuberculosis-mediated selection pressure much earlier, and more comprehensively, than was previously assumed.
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Box 1 | The pathology of the granuloma
Infection with Mycobacterium Blood vessel tuberculosis follows a relatively Infected alveolar well-defined sequence of macrophages events. The infectious bacilli are Airway inhaled as droplets from the Alveolar atmosphere. Exhaled droplets or macrophage nuclei are known to remain in Mycobacterium the atmosphere for several Mononuclear hours, and the infectious dose is cells estimated at a single bacterium. In the lung the bacteria are phagocytosed by alveolar macrophages and induce a localized proinflammatory response that leads to the Infected macrophage Foamy macrophages recruitment of mononuclear Foamy macrophages cells from neighbouring blood Lymphocyte Lymphocytes vessels (see figure). These cells are the building blocks for the granuloma, or tubercle, which is the signature of tuberculosis. The granuloma consists of a kernel of infected macrophages surrounded by foamy macrophages and other mononuclear phagocytes, with a Macrophages mantle of lymphocytes in Fibrous cuff Blood association with a fibrous cuff of vessels Macrophage collagen and other extracellular matrix components that delineates the periphery of the structure (see figure). This tissue response typifies the containment phase of the Free mycobacteria infection in which there are no Necrotic, caseating overt signs of disease and the granuloma centre Airway host does not transmit the infection to others. In the later stages, the granuloma develops a marked fibrous sheath and the Granuloma number of blood vessels penetrating the structure diminishes markedly. Histological studies with nitroimidazole indicate that at least the boundary to the central region of these granulomas is hypoxic30. Containment usually fails when the immune status of the host changes, which is usually a consequence of old age, malnutrition or co-infection with HIV basically any condition that reduces the number, or impairs the function, of CD4+ T cells. Following such a change in immune status the granuloma caseates (decays into a structureless mass of cellular debris), ruptures and spills thousands of viable, infectious bacilli into the airways. This results in the development of a productive cough that facilitates aerosol spread of infectious bacilli.

Although M. tuberculosis can cause primary disease, most notably in neonates, the most common manifestation of TB in adults is the reactivation of a pre-existing, chronic infection. Reactivation is frequently triggered by conditions that compromise immune surveillance, including AIDS, poor nutrition, old age and stress, all conditions that would seem to impact primarily on a post-breeding-age population. But this is a modern interpretation and it is likely that, under the more extreme living conditions to which prehistoric man was exposed, progression to fullblown, primary disease was more common and would

have impacted on human reproduction. If the human population ran the gauntlet of such selection pressure en masse as it emerged from Africa, this could have contributed to the relatively low incidence of marked TB susceptibility traits. Although reactivation and disease progression are usually thought of as a failure of the immune system, this is an extremely humanocentric interpretation that denies the key position that transmission occupies in the life cycle of M. tuberculosis. This article examines the active role of M. tuberculosis in the biology of the TB granuloma.

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Box 2 | The cytokine/chemokine storm
The recruitment of successive waves of cells to the forming granuloma is driven by a bewildering array of cytokines and chemokines that is initiated by the infected macrophage. Tumour necrosis factor (TNF)- is undoubtedly the dominant cytokine in this response and it elevates the production of the chemokines that mediate recruitment (see figure). Infected macrophages produce TNF-, and the chemokines CCL2 and CXCL10. In experimental bead granulomas there is a transient recruitment of neutrophils before the sustained migration of macrophages but it is unclear if this occurs in the natural infection where the insult is less. Analysis of broncholavage fluid and serum from infected individuals indicates that there is the production of a barrage of chemokines, including CCL2, CXCL10, CCL3,4,5 and CXCL9. Finally, in vivo studies of both infected mice and macaque monkeys have revealed extensive upregulation of the CXCR3-binding cytokines CXCL9, CXCL10 and CXCL11 (REFS 92,93). These chemokines lead to the recruitment of lymphocytes including first natural killer (NK) T cells, then CD4+, CD8+ and T cells and B cells. The quietening down of the response coincides with the production of interferon (IFN)- (see figure). It is interesting to note that IFN--deficient mice are actually unable to turn off this proinflammatory cascade and suffer fatal pathology in their lungs94.

Infected macrophage TNF- (acts as a positive feedback loop) TNF-, CCL2, CXCL10, IL1-, IL1-, ILI8 Neutrophils

Caseation
The process by which a tuberculous granuloma decays into a structureless mass of cellular debris.

The cytokine/ chemokine storm

TNF-, CCL2, CXCL10, CXCL9, CCL5, CCL3, CCL4 Uninfected macrophages

Chemokine
Cytokines involved in specific inflammatory responses. They are differentiated into CC or CXC chemokines on the basis of their primary sequence.

Expands TNF production Lymphocytes (CD4+, CD8+ and T cells and B cells) IFN-

Natural killer (NK) T cell

An NK T cell is a T cell that expresses some NK cell receptors and has some NKcell-like functions. They also express a T-cell receptor that recognizes CD1b (which binds glycolipids not peptides).

Downregulates proinflammatory response

CD4+ cell
A subpopulation of T cells that express the CD4 receptor and respond to antigens presented on the surface of host cells that bear major histocompatibility complex class II molecules. Two distinct subsets of activated CD4+ T cells have been described. T-helper 1 (TH1) cells produce interferon , tumournecrosis factor and interleukin (IL-)12, and support cellmediated immunity. TH2 cells produce IL-4, IL-5 and IL-13, support humoral immunity, and downregulate TH1 responses.

CD8+ cell
A subpopulation of T cells that express the CD8 receptor. CD8+ cells recognize antigens that are presented on the surface of host cells by major histocompatibility complex class I molecules, leading to their destruction, and are therefore also known as cytotoxic T cells.

Cytokine
Member of a large family of secreted proteins that bind immune cells through specific receptors. Cytokine production results in the activation of an intracellular-signalling cascade that commonly regulates processes such as immune function and inflammation.

The development of the TB granuloma Most experimental infections conducted with M. tuberculosis use the murine host. The mouse is a useful tool for revealing the bacterial factors that are important in the infection process, and for probing the antigenic and cellular basis of a protective immune response. However, it is seriously compromised as a model for granuloma formation and progression. Mice do not build the highly stratified structures that are observed in humans and do not normally exhibit necrosis and caseation, which are significant factors leading to transmission (BOX1). Retrospective histological and molecular studies of TB infection in humans have yielded invaluable insights into the progression of the disease, especially when interpreted in the context of in vitro murine macrophage studies. In vitro analyses of the responses of murine and human macrophages to M. tuberculosis infection indicate that the cells produce a robust proinflammatory response through the activity of Toll-like receptor (TLR) agonists (stimulators of the hosts TLRs) that are abundant on the surface of the bacteria. It is widely postulated that alveolar macrophages in the airways, following internalization of inhaled bacteria, are stimulated to invade the lung epithelium1820. Production of tumour necrosis factor (TNF)- and inflammatory chemokines from the infected macrophages drives the recruitment of successive waves of neutrophils, natural killer (NK) T cells, CD4+ T cells and CD8+ T cells, each of which produce their own complement of chemokines and cytokines that amplify cellular recruitment and remodelling of the infection site18,19,21. This inflammatory cascade (BOX 2) is regulated and superceded by a specific, cellular immune response that is linked to the production of interferon (IFN)-. At

this stage, the formation of the stable granuloma that is responsible for immune containment during the latent, or subclinical, period of the infection becomes recognizable and the stratification of the structure emerges2226. These studies have been invaluable in defining the overall trends observed in infected tissue. This, however, is the traditional view of granuloma progression and its tenets are being questioned by temporal studies in macaque monkeys that indicate a more heterogeneous picture in which caseation is observed even in early lesions, and there is marked heterogeneity between the granulomas present in a single host27. More mature-phase granulomas show marked neovascularization and develop an extensive fibrotic capsule that delineates the margin between the macrophages, granulocytes, foamy macrophages and giant cells, and the lymphocytic infiltrate18,25,26. In the late stage, the centre of the granuloma loses its vascular appearance and becomes necrotic. In a progressive lesion (BOX 1), the necrosis precedes and probably facilitates caseation, the granuloma wall breaks down and the bacteria are released into the airways, resulting in transmission. Transmission seems to coincide with high levels of matrix metalloproteinase 9 (MMP9) in the serum, implying that tissue breakdown is an active, destructive process. Recent histological analyses of human granulomas have been particularly informative in understanding the biology of these later structures18,22,25. The analysis of active cavitary lesions, compared with non-progressive tuberculomas, indicated that the non-progressive lesions were more highly vascularized, which is consistent both with the vascular epithelial growth factor (VEGF) production that is reported in activated macrophages28,29,

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and the accepted notion that progression to caseation occurs in a low-oxygen environment30. IFN--producing T cells were found in both progressive and non-progressive granulomas but were located predominantly outside the necrotic centre and the fibrous wall. One of the more intriguing observations in these studies was the relative distribution of bacteria and bacterial products. Several studies noted that, although bacteria were found in the central, necrotic region of the lesion, a significant portion of bacteria (or bacterial antigens) were associated with macrophages in the peripheral leukocytic infiltrate. These macrophages have been found both bordering the necrotic region and outside the fibrotic capsule. These regions are the sites of most lymphocyte proliferation. Intriguingly, the bacteria found in these peripheral macrophages were positive for the expression of ICL1 (isocitrate lyase)23,31, which is known to be upregulated by M. tuberculosis in macrophages that are exposed to activating cytokines32. These observations indicate that the interactions at the periphery of the granuloma are dynamic. The human tuberculosis granuloma is the product of a robust cellular immune response to bacterial components; it is not by chance that the dead mycobacteria are used as the active ingredient in Freunds complete adjuvant. In AIDS patients, the diminished capacity to mount a CD4+ T-cell response correlates with reduced granuloma-forming capacity and, significantly, a reduced ability to prevent metastasis of infection. Most monographs detailing granuloma structure and function deal with this immune-mediated containment of infection and tend to treat transmission as a breakdown in immune function, but the bacterium has a vested interest in driving transmission and its capacity to divert the hosts response must be considered in greater depth. In short, how does M. tuberculosis influence this process to maximize its survival and subsequent transmission under strong immune pressure? Modulation of the phagosome seems to be mediated by both cell-wall lipids and other bacterial effectors33,35. Mutants that are partially defective in phagosome modulation reside in vacuoles of pH 5.8, which arrests bacterial growth39. In addition, immune activation of the macrophage enables it to overcome the influence of the bacterium on phagosome maturation and deliver it to a more acidic (pH 5.2) bactericidal environment. The fusion of the Mycobacterium-containing phagosome with lysosomes is achieved through the activity of the IFN-inducible p47 GTPase family4042. These data imply that the growth status of the bacteria can be impacted by the immune modulation of their host macrophages to induce either growth arrest or death. Given the differential distribution of intracellular and extracellular bacteria observed in human granulomas it is interesting to speculate on which bacteria are capable of replication: those in the necrotic core that are thought to be hypoxic, or those at the periphery in macrophages that are accessible to the activating cytokines produced by proliferating lymphocytes. Extreme activation of host macrophages in vitro by cytokines or through the induction of autophagy will result in bacterial death. However, in vivo, it is more likely that the difference between activated and resting macrophages is a continuum that determines bacterial growth rates rather than mediating clearance. This conclusion is supported by two studies that examined the stability of the bacterial population in infected mice and failed to detect an accumulation of bacterial debris, at both the histological and molecular levels43,44. Because the relative number of genome equivalents was stable and no accumulation of bacterial skeletons was observed, both studies concluded that there was a relatively stable population of bacteria, probably non-replicative, during chronic infection.

Neovascularization
The formation of new blood vessels in a developing tissue. This process is stimulated by the production of vascular endothelial growth factor. The term is used most frequently in cancer biology in which the tumour develops its own blood supply through neovascularization.

Foamy macrophage
A macrophage loaded with lipid droplets. Such cells are often observed in tissues with chronic proinflammatory stimulus.

Giant cell
A giant, multinucleate macrophage.

Tuberculoma
The tuberculoma is the granuloma that is formed during tuberculosis infection. This term is most frequently used by clinicians and has replaced the more traditional tubercle.

Phagosome
A membrane-bound cytoplasmic vacuole formed around a particle ingested by phagocytosis.

Homotypic fusion
The fusion of identical compartments or vesicles.

Autophagy
A pathway for the recycling of cellular contents, in which materials inside the cell are packaged into vesicles and are then targeted to the vacuole or lysosome for bulk turnover.

Survival inside macrophages Pathogenic mycobacterial species survive inside macrophages by arresting the normal maturation of their phagosome, thereby restricting its acidification to pH 6.4 and limiting fusion with pre-formed lysosomes3335. The characteristics of this compartment and its intersection with cellular membrane trafficking pathways are illustrated in FIG. 1. The strategy adopted by M. tuberculosis limits the hostility of the intracellular environment. However, the Mycobacterium-containing vacuole is not isolated or inert. It communicates with the extracellular environment by homotypic fusion with other compartments in the early recycling endosomal system36. The accessibility of the vacuole was demonstrated by the rapidity with which the vacuole acquired GM1 ganglioside from the macrophage plasmalemma37. These data were all generated in infected macrophages in culture. Although it still falls short of a true in vivo analysis, it has been shown that M. tuberculosis-containing vacuoles in infected macrophages isolated directly from broncholavage fluid from TB patients fail to acidify and do not acquire lysosomal cargo38.

Beyond phagocytes The development of the granuloma is clearly mediated by different populations of host cells and is absolutely dependent on the presence of a robust cellular immune response directed against bacterial antigens. However, it remains in the best interests of the bacterium to manipulate this response locally to ensure persistence and, ultimately, late stage damage and transmission. There is a rich literature dating back to the 1960s that identified the potency of a range of mycobacterial cell-wall lipids in the manipulation of immune response, adjuvanticity and the induction of granulomas4554 (and therefore their use in Freunds complete and Ribi adjuvant). Despite this fascinating set of early observations, our appreciation of how bacterial components might function as effectors that promote the success of the bacterium are only now emerging. Previous studies revealed that the M. tuberculosis cell-wall components lipoarabinomannan and arabinomannan were released inside infected cells and trafficked to compartments that lacked bacteria55. Further analysis of this process using M. bovis bacille CalmetteGurin (BCG) cells that had their surface glycoconjugates tagged with Texas Red hydrazide revealed that the mycobacterial constituents were associated with several intracellular organelles but accumulated in the internal vesicles in lysosomes56 (FIG. 2).

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a
7.5 7.0 6.5

Mycobacterium

6.0 5.5 5.0 4.5 0 5 10 15 20 25 30

IgG-beads

Time (min)

b
IgG-coated bead

complex (MHC) class II-enriched compartments (MIIC) or multi-vesicular lysosomes (FIG. 2). These mycobacterial lipids could also be observed in uninfected bystander cells56. The release of these vesicles seemed to occur through a constitutive form of exocytosis57. Clearly, if these lipids possessed biological activities then their transfer to bystander cells would expand the bacterias sphere of influence beyond the immediate confines of the host cell. Subsequent studies have shown that similar vesicles containing bacterial components can be generated through the apoptotic death of infected macrophages and dendritic cells58,59, and that bacterial lipids might be released and transferred between cells as complexes with apolipoprotein60, although it is unclear if all these pathways are functionally equivalent.

pH

Early endosome Nucleus pH 4.8

Lysosome

pH 6.4 Transferrin Recycling pathway

Transferrin Early endosome with transferrin Mycobacterium

Figure 1 | Trafficking of Mycobacterium tuberculosis bacilli in cells. Following phagocytosis, phagosomes containing IgG-coated beads acidify rapidly to pH 5 (a) and lower, and the inert particles are subsequently delivered to the lysosome (b). By contrast, phagosomes containing M. tuberculosis only acidify to pH 6.4 (a) and fail to fuse with lysosomes. The vacuoles containing M. tuberculosis retain many of the characteristic of the early endosomal system (b). They are accessible to recycling endosomes, as evidenced by their acquisition of labelled transferrin when it is added to the external medium. Components of the peripheral cell wall of the bacteria that are shed into the bacteria-containing vacuoles traffic out of the vacuoles and coalesce in dense, lysosomal compartments. Finally, the bacteria-containing vacuoles, in common with most endosomal/lysosomal stages, fuse with delivery vesicles trafficking from the trans-Golgi network of the host cell. The data imply that the vacuoles in which M. tuberculosis reside are not particularly hostile with respect to both pH and hydrolytic activity35.

The bioactive lipids of M. tuberculosis Analysis of the lipid species present in the exosomal fraction identified the main peripheral lipids from the bacterial cell wall56,61. This implies that release is passive, or at least non-selective, possibly mediated by the surfactant environment in the endosomal system. Characterization of the lipids by thin-layer chromatography, differential dye labelling electrospray ionization mass spectrometry and collisionally activated tandem mass spectrometry led to the determination that the main released lipids corresponded to phosphatidylinositol mannoside species 2A and 2B, monophosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, trehalose mycolates and the phenolic glycolipid mycoside B. Mycobacterial cell-wall-associated proteins belonging to the Ag85 family were also detected in the exosomes from infected macrophages, supporting the view that this is a mass-action effect62. Although it has not been documented in the same detail, it is likely that all released, hydrophobic components from the bacteria will follow a similar pathway through, and out of, the infected macrophage. Antigens versus effectors Many of these released bacterial components are known immunomodulators, but if they traffic with the released bacterial lipids they will aggregate in the multivesicular lysosome, or the MIIC, of the host cell. Whereas the M. tuberculosis-containing vacuole itself is sequestered outwith the antigen presenting machinery of the cell63, the bacterial components that are released are fodder for the antigen-presenting machinery and become antigens that could be recognized on the surface of the infected cell. Protein antigens secreted into the culture filtrate by growing bacteria in culture have been shown to include some of the more immunogenic bacterial proteins, such as early secreted antigenic target 6 (ESAT6) and culture filtrate protein 10 (CFP10) (REFS 6466), and several of the lipids can be presented by the CD1 family of MHC molecules6769. Furthermore, the antigen-loaded vesicles from infected cells are capable of mediating cross-priming58,59. These released antigens could therefore have a role in driving the proliferative response observed at the periphery of the granuloma.
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The released bacterial constituents traffic through the infected host cell and, significantly, were also present in an extracellular microvesicular fraction56,57. Further analysis of the exosomal vesicles revealed the presence of lysosomal membrane proteins and lysosomal proteases, indicating that labelled mycobacterial cell-wall constituents were released from a lysosomal compartment. Ultrastructural analyses of infected cells showed that the bacterial lipids accumulated in multilamellar vesicles that were morphologically reminiscent of major histocompatibility

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Fibrosis
Fibrosis is frequently seen at sites of chronic inflammatory stimulation. Cells lay down a fibrinogen/fibrin skeleton that is augmented with other extracellular matrix proteins like collagen.

Some lipids are more equal than others The identification of mycobacterial cell-wall lipids that are involved in the induction or control of the disease pathology that promotes bacterial survival has proceeded predominantly through direct analysis of the biological activities of individual, purified bacterial cell-wall components. So although the data documenting the release and trafficking of bacterial lipids inside their host cell are recent, there is a rich history of publications on the biomodulatory activities of mycobacterial lipids. In the 1960s, researchers became interested in the capacity of defined bacterial cell-wall constituents to induce granulomatous responses in mice52,53. Since these seminal studies, several laboratories have explored the effects of isolated bacterial cell-wall lipids on macrophages

Nucleus

a
Lipids are shed Mycobacterium tuberculosis

Lipids accumulate in multilamellar bodies

Multilamellar bodies coalesce in multivesicular lysosome

Lipids released in exosomes from cell surface

Spread to bystander cells

Figure 2 | Release and trafficking of bacterial lipids. Mycobacterium tuberculosis sheds its peripheral cell-wall components into its vacuole. These lipids accumulate in multilamellar bodies (a) in mixed micellar structures that contain both host- and pathogen-derived components. The multilamellar bodies coalesce in the multivesicular lysosome, known as the major histocompatibility complex class II-enriched compartment in antigen-presenting cells (b). The vesicles gain egress from the infected macrophage by exocytosis and are released as exosomes into the external milieu (c). The exosomes, carrying pathogen-derived lipids and proteins, are internalized by neighbouring cells (d)56,57,62.

and T cells in culture70,71, and in mice7277. The relevance of these data to the tuberculoma is difficult to extrapolate because the in vitro experiments were done using select cell types (usually macrophages) and might lack key players, whereas the in vivo experiments are complex and have limited accessibility for analysis and manipulation. Among the in vivo experiments are a few studies that have localized the tissue response either through the use of particulate delivery systems for bacterial components74,78,79 or restriction to a specific site, such as the use of the air-pouch model76; both of these methods facilitate analysis at specific foci. Trehalose dimycolate (TDM) incorporated into oil droplets45,80 or on particles75,78,79 induces strong granulomatous reactions at sites of accumulation. More recently, lipid-coated particles that were incorporated into an extracellular matrix gel were inoculated into mice to study the waves of cellular recruitment and tissue remodelling induced by the bacterial lipids79. Fluorescently tagged lipids were internalized by macrophages and newly recruited inflammatory cells, and these cells generated a proinflammatory response that involved TNF-, interleukin (IL)-1, IL-6, IL-10, CCL2 and IFN--inducible protein-10 (CXCL10). Bacteriallipid-bearing matrices recruited greater numbers of inflammatory cells than control matrices that contained phosphatidylglycerol or no lipid. Histological characterization (FIG. 3) indicated that leukocytes arrived by two routes, either adherence to the surface and migration into freely floating matrices or extravasation directly from the circulation into newly vascularized matrices. Leukocytes arrived in successive waves: neutrophils, followed by macrophages and mature dendritic cells, and finally T cells, NK cells and B cells. This model aptly demonstrates the inflammatory properties of peripheral BCG cell-wall lipids, reproducing cellular and cytokine responses that are typical of granulomas in Mycobacterium-infected tissues. In addition the structure also reveals that many of the structural characteristics of TB granulomas (a capsular structure, extensive fibrosis and neovascularization) were recapitulated in this artificial model79. The similarities imply that the bacterial lipids have a significant role in shaping the tissue response at the infection site. The in vivo granuloma model was used to assay the bioactivity of the individual lipid species with respect to cell recruitment and cytokine induction. BCG-derived cell-wall lipids were fractionated and the isolated fractions, including phosphatidylinositol dimannosides, cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, trehalose monomycolate, TDM and mycocide B were assayed for activity61,78. Trehalose mono- and dimycolates were found to induce profound recruitment of a range of immune effector cells in vivo, indicating that the trehalose mycolates, particularly TDM, are the most bioactive lipids in the mycobacterial cell-wall fraction78. The potency of the response to TDM in comparison to other mycobacterial lipids is clear even at the histological level. These data are consistent with the rich body of literature documenting the bioactivity of trehalose mycolates both in vitro and in vivo51,61,71,72,8183.

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Fibrous layer

Fat cells at base of peritoneal cavity Neovascularization

Blood vessels Polystyrene bead coated with TDM

Matrigel matrix Lymphocyte cuffing Cells around the beads

Figure 3 | The lipid-bead granuloma model. Histological examination of lipid-bead granulomas reveals many of the features that are observed in human tuberculosis granulomas. The lipid-coated beads are clearly visible in the Matrigel matrix. The beads are surrounded by cells recruited into the structure. The granulomatous structure shows extensive neovascularization and marked lymphocyte cuffing around the new blood vessels. Finally there is extensive fibrotic remodelling of the tissue. This is most marked at the basement margin where the granuloma adheres to the fat cells in the peritoneal cavity. TDM, trehalose dimycolate.

Signature-tagged mutagenesis
A technique to screen large numbers of distinct mutants for those that fail to survive an animal infection. Each mutant is tagged with a unique DNA sequence (called a signature tag), which allows a specific mutant to be tracked within a large pool of bacteria.

Genetic screens for cell-wall lipid functions The use of genetics to probe the biological activity of the cell-wall lipids is problematic, owing to the restricted number of mutants available because the major classes of lipids seem to be essential. However, three lipid modulators have been analysed by mutational approaches, including phthiocerol dimycocerosate (PDIM), TDM and a phenolic glycolipid. Although the infection studies have been carried out predominantly in mice, these data are probably significant to the regulation of pathology at the infection site in humans. The activity of TDM in modulating the host inflammatory response has been confirmed and extended by mutational studies that revealed that TDM is modified by an unusual cyclopropane addition. Mutants defective in either the production of cyclopropane or the addition of cyclopropane to TDM have a markedly enhanced inflammatory response at a comparable bacterial load in murine infection experiments8486. This in vivo pathology translated into a five-fold increase in the level of TNF-

released by infected macrophages in vitro. These results are intriguing because they imply that the pathological properties of the lipid have been designed to mediate a level of damage appropriate to the survival and persistence strategies of the bacteria. Signature-tagged mutagenesis screens were used to identify loci involved in the synthesis and export of PDIM, which resulted in a growth defect in macrophages and in mice8789. Interestingly, the growth defect of the mutated M. tuberculosis strains in mice seemed to be tissue specific, and was restricted to the lung whereas bacterial loads in the liver and spleen were unaffected. The authors hypothesized that the lipid had a role in regulating the immune response within the infected tissue site89, although more work is needed to elucidate the tissue-dependency of the phenotype. Finally, it has long been known that the W-Beijing family of human TB isolates are particularly virulent in humans, and induce extreme pathology at a relatively low bacterial load in mice. Analysis of bacterial glycolipids in mice and rabbit models identified a phenolic glycolipid, similar to PDIM, that was unique to these W-Beijing strains90,91. Deletion of genes in a locus that encodes the machinery for polyketide and PDIM synthesis led to loss of this lipid and a marked reduction in the hypervirulence phenotype. Analysis of the response of macrophages to purified phenolic glycolipid indicated that the lipid suppressed their proinflammatory response, resulting in depressed production of TNF-, IL-6, IL-12 and CCL2. How this translates into virulence in vivo remains to be determined but the data reciprocate the theme that virulence and pathology must be regulated to yield a pathogen capable of chronicity, reactivation and transmission. Clearly, a genetic route in generating bacterial mutants with structurally defined defects in their cell-wall constituents is attractive. The data emerging from such studies are tantalizing but they underline the complexity of the interaction and emphasize the divide between macrophage experiments in vitro and the results of in vivo challenge in a multicellular environment.

Transmission This Review has attempted to generate a picture of a strong host immune response that provides the building blocks that form the granulomatous tissue at the infection site while emphasizing that the arrangement of these blocks is clearly influenced by effectors released by M. tuberculosis. It is relatively easy to envisage how the inflammatory cell-wall components of M. tuberculosis can aggravate its host into a strong proinflammatory response that causes the tissue to compartmentalize this persistent irritant behind a fibrous capsule. Artificial models with isolated lipids arrayed on particles reciprocate many of the characteristics of true tubercles during their early stages of development. The real challenge, however, is to understand what the bacterium can do to drive the late-stage damage that leads to transmission. TDM is an interesting candidate for such a mediator. It seems
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to have evolved structurally to induce a measured degree of pathology85,86, and its biological activity is regulated by its mode of presentation73,78,80. The recent observation that TDM associates with lipid droplets in necrotic regions of murine granulomas80 is intriguing because it provides a potential means of enhancing the activity of TDM in the late-stage granulomas, allowing it to be at its most destructive when damage is needed to drive transmission. In earlier in vivo experiments, the toxicity of TDM was enhanced markedly by its incorporation into oil droplets or large particles45,51,73,78. Profiling the transcriptional response in defined areas of human TB granulomas through the use of microarray analysis of laser-capture micro-dissected regions from human tissue might provide some insights into the pathways activated in the host during this crucial transition. The application of this information to more appropriate animal models such as guinea pigs, rabbits or monkeys might even allow these pathways to be probed experimentally. Although it might be a pipedream to imagine we could ever block the transmission of M. tuberculosis, we should at least aim to understand how the propagation of this successful pathogen is achieved.

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Acknowledgements
This work was supported by grants from the National Institute of Allergy and Infectious Diseases and the National Heart, Lung and Blood Institute of the National Institutes of Health, USA. The author would like to acknowledge the work of past and present members of the laboratory, most notably E. Rhoades, R. Geisel and K. Sakamoto.

Competing interests statement

The author declares no competing financial interests.

DATABASES
The following terms in this article are linked online to: Entrez Genome Project: http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi?db=genomeprj Mycobacterium bovis | Mycobacterium tuberculosis

FURTHER INFORMATION
David G. Russells homepage: http://www.vet.cornell.edu/ public/microbiology/russellnew2.htm Access to this links box is available online.

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