Russell (2006) Tubercle

7/30/2019 Russell (2006) Tubercle

http://slidepdf.com/reader/full/russell-2006-tubercle 1/9

The bacterium Mycobacterium tuberculosis is the causa-tive agent of one of those human conditions that, despitethe developments of modern medicine, is reluctant torelinquish its grip on mankind. The incidence of tuber-culosis (TB) correlates strongly with reduced socio-eco-nomic status, thereby adding to the burden of the globalpoor1,2. Estimates of host penetrance based on skin-testconversion indicate that up to one-third of the world’spopulation are infected with this bacterium. Over theirlifetime it is projected that an infected individual has a5–10% risk of developing TB3, although this scenarioshows extraordinary regional variation that is impactedto a tragic extent by HIV infection4. Given that such arelatively low number of individuals develop disease andtherefore the capacity to transmit the infection, it fol-lows that the transmission process must be supremely efficient to sustain such levels of infection in its hostpopulation.

The progression of the disease is determined at thelevel of the infection site itself. Once the bacterium hasgained entry into a macrophage and triggered that hostcell to invade the tissue of the lung, the host respondsby remodelling the site of infection into a cellular mass,

the ‘tubercle’ or granuloma that has given the disease itsname. The human tuberculosis granuloma is a beautifuland fascinating structure in which all the acts of the dis-ease are written and performed, as documented in BOX 1.The bulk of the literature on the tuberculosis granulomafocuses on the host component and ignores any influ-ence that the bacterium might have on its progression.However, given that transmission is key to the successof the bacterium, it would be short-sighted to imaginethat M. tuberculosis has not evolved specific strategiesand effectors to maximize progression to a productiveinfection. This Review attempts to redress the balancein favour of M. tuberculosis.

What is now ancient historyTo appreciate the biological significance of the granu-loma it is important to consider tuberculosis in thecontext of human evolution rather than focusing exclu-sively on the current manifestations of the disease. How has TB impacted on mankind through pre-history?Until recently, it was thought that M. tubercu losis evolved from Mycobacterium bovis and was acquiredby humans during the development of agriculture inthe Fertile Crescent around 9,000 years ago5, a hypoth-esis popularized in the book Guns, Germs and Steel by Jared Diamond6. In this scenario, TB would have beenintroduced into a subpopulation of mankind after theiremergence from Africa and one would have predicted,unless other infectious diseases provided comparableselective pressures on fitness and reproduction, thatthe current human populations would exhibit extremetraits of susceptibility However, this is not the case— with the exception of a few extremely rare mutationsin central immune system function7, the populationgenetics of susceptibility to TB reveal relatively mod-est associations of individual genotypes with diseaseprogression7–13.

More recent studies into the evolution of M. tubercu-losis have indicated that M. tuberculosis is actually moreclosely related to a common founder strain than it is to M. bovis. This implies that either humans infected cat-tle with M. tuberculosis, resulting in the divergence of M. bovis , or the two strains evolved in parallel from afounder strain that infected both humans and cattle14–16.The ancestor of M. tuberculosis most likely arose fromthe M. tuberculosis-like species of the M. tuberculosis complex that are found today in central Africa14–17. Thismight indicate that humans were exposed to tuberculo-sis-mediated selection pressure much earlier, and morecomprehensively, than was previously assumed.

Department of Microbiology

and Immunology, College of

Veterinary Medicine, Cornell

University, Ithaca, New York

14853, USA.

e-mail: [email protected]

doi:10.1038/nrmicro1538

Published online

11 December 2006

Founder strain

The ancestral species or strain

that underwent divergent

evolution to produce several

new species or strains.

Who puts the tubercle in tuberculosis?David G. Russell

Abstract | Tuberculosis (TB), an illness that mainly affects the respiratory system, is one of the

world’s most pernicious diseases. TB currently infects one-third of the world’s 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 granuloma’s 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.

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NATURE REVIEWS | MICROBIOLOGY VOLUME 5 | JANUARY 2007 | 39



Airway

Free mycobacteria

Necrotic, caseatinggranuloma centre

Granuloma

Macrophage

LymphocytesLymphocyte

Infected macrophage

Airway

Mycobacterium

Blood vessel

Infected alveolarmacrophages

Mononuclearcells

Alveolarmacrophage

Foamy macrophages

Fibrous cuff Macrophages

Foamy macrophages

Bloodvessels

Although M. tuberculosis can cause primary dis-ease, most notably in neonates, the most commonmanifestation of TB in adults is the reactivation of a pre-existing, chronic infection. Reactivation isfrequently triggered by conditions that compromiseimmune surveillance, including AIDS, poor nutrition,old age and stress, all conditions that would seem toimpact 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 whichprehistoric man was exposed, progression to full-blown, primary disease was more common and would

have impacted on human reproduction. If the humanpopulation ran the gauntlet of such selection pressureen masse as it emerged from Africa, this could havecontributed to the relatively low incidence of markedTB susceptibility traits.

Although reactivation and disease progression areusually thought of as a ‘failure’ of the immune system,this is an extremely humanocentric interpretation thatdenies the key position that transmission occupies in thelife cycle of M. tuberculosis. This article examines theactive role of M. tuberculosis in the biology of the TBgranuloma.

Box 1 | The pathology of the granuloma

Infection with Mycobacterium

tuberculosisfollows a relatively

well-defined sequence of

events. The infectious bacilli are

inhaled as droplets from the

atmosphere. Exhaled droplets or

nuclei are known to remain inthe atmosphere for several

hours, and the infectious dose is

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

recruitment of mononuclear

cells from neighbouring blood

vessels (see figure). These cells

are the building blocks for the

granuloma, or tubercle, which is

the signature of tuberculosis.

The granuloma consists of akernel of infected macrophages

surrounded by foamy

macrophages and other

mononuclear phagocytes, with a

mantle of lymphocytes in

association with a fibrous cuff of

collagen and other extracellular

matrix components that

delineates the periphery of the

structure (see figure). This tissue

response typifies the

‘containment’ phase of the

infection in which there are no

overt signs of disease and the

host does not transmit theinfection to others. In the later

stages, the granuloma develops

a marked fibrous sheath and the

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.

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Infectedmacrophage

Uninfected macrophages

TNF-α, CCL2, CXCL10, IL1-α, IL1-β, ILI8

TNF-α, CCL2, CXCL10, CXCL9,CCL5, CCL3, CCL4

TNF-α(acts as a

positive

feedbackloop)

The cytokine/chemokine storm

ExpandsTNF

production

Downregulatesproinflammatoryresponse

IFN-γ

Lymphocytes(CD4+, CD8+ and T cells andB cells)

Neutrophils

Caseation

The process by which a

tuberculous granuloma decays

into a structureless mass of

cellular debris.

Chemokine

Cytokines involved in specific

inflammatory responses. They

are differentiated into CC or

CXC chemokines on the basis

of their primary sequence.

Natural killer (NK) T cellAn NK T cell is a T cell that

expresses some NK cell

receptors and has some NK-

cell-like functions. They also

express a T-cell receptor that

recognizes CD1b (which binds

glycolipids not peptides).

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 γ , tumour-

necrosis factor α and interleukin

(IL-)12, and support cell-

mediated 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 complexclass 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 granulomaMost experimental infections conducted with M. tuber-culosis use the murine host. The mouse is a useful toolfor revealing the bacterial factors that are important inthe infection process, and for probing the antigenic andcellular basis of a protective immune response. However,it is seriously compromised as a model for granulomaformation and progression. Mice do not build the highly stratified structures that are observed in humans anddo not normally exhibit necrosis and caseation, whichare significant factors leading to transmission (BOX1).Retrospective histological and molecular studies of TBinfection in humans have yielded invaluable insightsinto the progression of the disease, especially wheninterpreted in the context of in vitro murine macrophagestudies. In vitro analyses of the responses of murine andhuman macrophages to M. tuberculosis infection indicatethat the cells produce a robust proinflammatory responsethrough the activity of Toll-like receptor (TLR) agonists(stimulators of the host’s TLRs) that are abundant on

the surface of the bacteria. It is widely postulated thatalveolar macrophages in the airways, following inter-nalization of inhaled bacteria, are stimulated to invadethe lung epithelium18–20. Production of tumour necrosisfactor (TNF)-α and inflammatory chemokines from theinfected macrophages drives the recruitment of succes-sive waves of neutrophils, natural killer (NK) T cells, CD4+

T cells and CD8+ T cells, each of which produce their owncomplement of chemokines and cytokines that amplify cellular recruitment and remodelling of the infectionsite18,19,21. This inflammatory cascade (BOX 2) is regulatedand superceded by a specific, cellular immune responsethat is linked to the production of interferon (IFN)-γ. At

this stage, the formation of the ‘stable’ granuloma that isresponsible for immune containment during the latent,or subclinical, period of the infection becomes recogniz-able and the stratification of the structure emerges22–26.These studies have been invaluable in defining the overalltrends observed in infected tissue. This, however, is thetraditional view of granuloma progression and its tenetsare being questioned by temporal studies in macaquemonkeys that indicate a more heterogeneous picture inwhich caseation is observed even in early lesions, andthere is marked heterogeneity between the granulomaspresent in a single host27.

More mature-phase granulomas show marked neo-

vascularization and develop an extensive fibrotic capsulethat delineates the margin between the macrophages,granulocytes, foamy macrophages and giant cells, and thelymphocytic infiltrate18,25,26. In the late stage, the centre of the granuloma loses its vascular appearance and becomesnecrotic. In a progressive lesion (BOX 1), the necrosis pre-cedes and probably facilitates caseation, the granuloma

wall breaks down and the bacteria are released into theairways, resulting in transmission. Transmission seemsto coincide with high levels of matrix metalloproteinase9 (MMP9) in the serum, implying that tissue breakdownis an active, destructive process.

Recent histological analyses of human granulomashave been particularly informative in understandingthe biology of these later structures18,22,25. The analysis of active cavitary lesions, compared with ‘non-progressive’tuberculomas, indicated that the non-progressive lesionswere more highly vascularized, which is consistent bothwith the vascular epithelial growth factor (VEGF) pro-duction that is reported in activated macrophages28,29,

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)-α is

undoubtedly the dominant cytokine in this response and

it elevates the production of the chemokines that mediate

recruitment (see figure). Infected macrophages produceTNF-α, 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 γδ T cells and

B cells. The quietening down of the response coincideswith the production of interferon (IFN)-γ (see figure). It is

interesting to note that IFN-γ -deficient mice are actually

unable to turn off this proinflammatory cascade and suffer

fatal pathology in their lungs94.

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NeovascularizationThe 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-boundcytoplasmic 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.

and the accepted notion that progression to caseationoccurs in a low-oxygen environment30. IFN-γ -producingT cells were found in both progressive and non-progres-sive granulomas but were located predominantly outsidethe necrotic centre and the fibrous wall.

One of the more intriguing observations in thesestudies was the relative distribution of bacteria andbacterial products. Several studies noted that, althoughbacteria were found in the central, necrotic region of the lesion, a significant portion of bacteria (or bacte-rial antigens) were associated with macrophages in theperipheral leukocytic infiltrate. These macrophageshave been found both bordering the necrotic regionand outside the fibrotic capsule. These regions are thesites of most lymphocyte proliferation. Intriguingly, thebacteria found in these peripheral macrophages werepositive for the expression of ICL1 (isocitrate lyase)23,31,which is known to be upregulated by M. tuberculosis inmacrophages that are exposed to activating cytokines32.These observations indicate that the interactions at theperiphery of the granuloma are dynamic.

The human tuberculosis granuloma is the productof a robust cellular immune response to bacterial com-ponents; it is not by chance that the dead mycobacteriaare used as the active ingredient in Freund’s completeadjuvant. In AIDS patients, the diminished capacity tomount a CD4+ T-cell response correlates with reducedgranuloma-forming capacity and, significantly, areduced ability to prevent metastasis of infection. Mostmonographs detailing granuloma structure and func-tion deal with this immune-mediated containment of infection and tend to treat transmission as a breakdownin immune function, but the bacterium has a vestedinterest in driving transmission and its capacity to divertthe host’s response must be considered in greater depth.In short, how does M. tuberculosis influence this processto maximize its survival and subsequent transmissionunder strong immune pressure?

Survival inside macrophagesPathogenic mycobacterial species survive inside mac-rophages by arresting the normal maturation of theirphagosome, thereby restricting its acidification to pH 6.4and limiting fusion with pre-formed lysosomes33–35. Thecharacteristics of this compartment and its intersectionwith cellular membrane trafficking pathways are illus-trated in FIG. 1. The strategy adopted by M. tuberculosis limits the hostility of the intracellular environment.

However, the Mycobacterium-containing vacuole is notisolated or inert. It communicates with the extracellularenvironment by homotypic fusion with other compart-ments in the early recycling endosomal system36. Theaccessibility of the vacuole was demonstrated by therapidity with which the vacuole acquired GM1 ganglio-side from the macrophage plasmalemma37. These datawere all generated in infected macrophages in culture.Although it still falls short of a true in vivo analysis,it has been shown that M. tuberculosi s-containing

vacuoles in infected macrophages isolated directly frombroncholavage fluid from TB patients fail to acidify anddo not acquire lysosomal cargo38.

Modulation of the phagosome seems to be mediatedby both cell-wall lipids and other bacterial effectors33,35.Mutants that are partially defective in phagosomemodulation reside in vacuoles of pH 5.8, which arrestsbacterial growth39. In addition, immune activation of themacrophage enables it to overcome the influence of thebacterium on phagosome maturation and deliver it toa more acidic (pH 5.2) bactericidal environment. Thefusion of the Mycobacterium-containing phagosomewith lysosomes is achieved through the activity of theIFN-inducible p47 GTPase family 40–42. These data imply that the growth status of the bacteria can be impactedby the immune modulation of their host macrophagesto induce either growth arrest or death.

Given the differential distribution of intracellular andextracellular bacteria observed in human granulomas itis interesting to speculate on which bacteria are capableof replication: those in the necrotic core that are thoughtto be hypoxic, or those at the periphery in macrophagesthat are accessible to the activating cytokines producedby proliferating lymphocytes. Extreme activation of host

macrophages in vitro by cytokines or through the induc-tion of autophagy will result in bacterial death. However,in vivo, it is more likely that the difference betweenactivated and resting macrophages is a continuum thatdetermines bacterial growth rates rather than mediatingclearance. This conclusion is supported by two studiesthat examined the stability of the bacterial populationin infected mice and failed to detect an accumulation of bacterial debris, at both the histological and molecularlevels43,44. Because the relative number of genome equiva-lents was stable and no accumulation of bacterial ‘skel-etons’ was observed, both studies concluded that therewas a relatively stable population of bacteria, probably non-replicative, during chronic infection.

Beyond phagocytesThe development of the granuloma is clearly mediated by different populations of host cells and is absolutely depend-ent on the presence of a robust cellular immune responsedirected against bacterial antigens. However, it remainsin the best interests of the bacterium to manipulate thisresponse locally to ensure persistence and, ultimately, latestage damage and transmission. There is a rich literaturedating back to the 1960s that identified the potency of arange of mycobacterial cell-wall lipids in the manipula-tion of immune response, adjuvanticity and the inductionof granulomas45–54 (and therefore their use in Freund’s

complete and Ribi adjuvant). Despite this fascinating setof early observations, our appreciation of how bacterialcomponents might function as effectors that promote thesuccess of the bacterium are only now emerging. Previousstudies revealed that the M. tuberculosis cell-wall com-ponents lipoarabinomannan and arabinomannan werereleased inside infected cells and trafficked to compart-ments that lacked bacteria55. Further analysis of this proc-ess using M. bovis bacille Calmette–Guérin (BCG) cellsthat had their surface glycoconjugates tagged with TexasRed hydrazide revealed that the mycobacterial constituentswere associated with several intracellular organelles butaccumulated in the internal vesicles in lysosomes56 (FIG. 2).

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Transferrin

Early endosome

pH 6.4

pH 4.8

Recycling pathway

Early endosomewith transferrin

Transferrin

Mycobacterium

Lysosome

Time (min)

Mycobacterium

IgG-beads

p H

a

b

04.5

5.0

5.5

6.0

6.5

7.0

7.5

5 10 15 20 25 30

IgG-coated bead

Nucleus

The released bacterial constituents traffic through theinfected host cell and, significantly, were also present in anextracellular microvesicular fraction56,57. Further analysisof the exosomal vesicles revealed the presence of lysosomalmembrane proteins and lysosomal proteases, indicatingthat labelled mycobacterial cell-wall constituents werereleased from a lysosomal compartment. Ultrastructuralanalyses of infected cells showed that the bacterial lipidsaccumulated in multilamellar vesicles that were mor-phologically reminiscent of major histocompatibility

complex (MHC) class II-enriched compartments (MIIC)or multi-vesicular lysosomes (FIG. 2). These mycobacte-rial lipids could also be observed in uninfected bystandercells56. The release of these vesicles seemed to occurthrough a constitutive form of exocytosis57. Clearly, if these lipids possessed biological activities then theirtransfer to bystander cells would expand the bacteria’ssphere of influence beyond the immediate confines of the host cell. Subsequent studies have shown that similar

vesicles containing bacterial components can be gener-ated through the apoptotic death of infected macrophagesand dendritic cells58,59, and that bacterial lipids mightbe released and transferred between cells as complexeswith apolipoprotein60, although it is unclear if all thesepathways are functionally equivalent.

The bioactive lipids of M. tuberculosis

Analysis of the lipid species present in the exosomal frac-tion identified the main peripheral lipids from the bacte-rial cell wall56,61. This implies that release is passive, or atleast non-selective, possibly mediated by the surfactant

environment in the endosomal system. Characterizationof the lipids by thin-layer chromatography, differentialdye labelling electrospray ionization mass spectrometry and collisionally activated tandem mass spectrometry led to the determination that the main released lipidscorresponded to phosphatidylinositol mannoside species2A and 2B, monophosphatidylglycerol, diphosphatidyl-glycerol, phosphatidylethanolamine, trehalose mycolatesand the phenolic glycolipid mycoside B.

Mycobacterial cell-wall-associated proteins belong-ing to the Ag85 family were also detected in the exo-somes from infected macrophages, supporting the view that this is a mass-action effect62. Although it has notbeen documented in the same detail, it is likely that allreleased, hydrophobic components from the bacteriawill follow a similar pathway through, and out of, theinfected macrophage.

Antigens versus effectorsMany of these released bacterial components are knownimmunomodulators, but if they traffic with the releasedbacterial lipids they will aggregate in the multivesicularlysosome, or the MIIC, of the host cell. Whereas the M. tuberculosis-containing vacuole itself is sequesteredoutwith the antigen presenting machinery of the cell63,the bacterial components that are released are fodderfor the antigen-presenting machinery and become

antigens that could be recognized on the surface of theinfected cell.

Protein antigens secreted into the culture filtrate by growing bacteria in culture have been shown to includesome of the more immunogenic bacterial proteins, suchas early secreted antigenic target 6 (ESAT6) and culturefiltrate protein 10 (CFP10) (REFS 64–66), and several of the lipids can be presented by the CD1 family of MHCmolecules67–69. Furthermore, the antigen-loaded vesiclesfrom infected cells are capable of mediating cross-prim-ing58,59. These released antigens could therefore have arole in driving the proliferative response observed at theperiphery of the granuloma.

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, phagosomescontaining 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

.

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Spread to bystander cells

Lipids released in exosomesfrom cell surface

Lipids are shed

Mycobacteriumtuberculosis

Nucleus

Lipids accumulatein multilamellar bodies Multilamellar bodies coalesce

in multivesicular lysosome

a

b

c

d

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 thatare involved in the induction or control of the diseasepathology that promotes bacterial survival has pro-ceeded predominantly through direct analysis of the bio-logical activities of individual, purified bacterial cell-wallcomponents. So although the data documenting therelease and trafficking of bacterial lipids inside their hostcell are recent, there is a rich history of publications on thebiomodulatory activities of mycobacterial lipids.

In the 1960s, researchers became interested in thecapacity of defined bacterial cell-wall constituents toinduce granulomatous responses in mice52,53. Since theseseminal studies, several laboratories have explored theeffects of isolated bacterial cell-wall lipids on macrophages

and T cells in culture70,71, and in mice72–77. The relevanceof these data to the tuberculoma is difficult to extrapolatebecause the in vitro experiments were done using selectcell types (usually macrophages) and might lack key play-ers, whereas the in vivo experiments are complex and havelimited accessibility for analysis and manipulation.

Among the in vivo experiments are a few studies thathave localized the tissue response either through theuse of particulate delivery systems for bacterial compo-nents74,78,79 or restriction to a specific site, such as the useof the air-pouch model76; both of these methods facilitateanalysis at specific foci. Trehalose dimycolate (TDM)incorporated into oil droplets45,80 or on particles75,78,79 induces strong granulomatous reactions at sites of accu-mulation. More recently, lipid-coated particles that wereincorporated into an extracellular matrix gel were inocu-lated into mice to study the waves of cellular recruitmentand tissue remodelling induced by the bacterial lipids79.Fluorescently tagged lipids were internalized by mac-rophages and newly recruited inflammatory cells, andthese cells generated a proinflammatory response that

involved TNF-α, interleukin (IL)-1α, IL-6, IL-10, CCL2and IFN-γ -inducible protein-10 (CXCL10). Bacterial-lipid-bearing matrices recruited greater numbers of inflammatory cells than control matrices that containedphosphatidylglycerol or no lipid.

Histological characterization (FIG. 3) indicated thatleukocytes arrived by two routes, either adherence tothe surface and migration into freely floating matricesor extravasation directly from the circulation into newly

vascularized matrices. Leukocytes arrived in succes-sive waves: neutrophils, followed by macrophages andmature dendritic cells, and finally T cells, NK cells andB cells. This model aptly demonstrates the inflammatory properties of peripheral BCG cell-wall lipids, reproduc-ing cellular and cytokine responses that are typical of granulomas in Mycobacterium-infected tissues. In addi-tion the structure also reveals that many of the structuralcharacteristics of TB granulomas (a capsular structure,extensive fibrosis and neovascularization) were recapitu-lated in this artificial model79. The similarities imply thatthe bacterial lipids have a significant role in shaping thetissue response at the infection site.

The in vivo granuloma model was used to assay the bioactivity of the individual lipid species withrespect to cell recruitment and c ytokine induction.BCG-derived cell-wall lipids were f ractionated andthe isolated fractions, including phosphatidylinositol

dimannosides, cardiolipin, phosphatidylglycerol, phos-phatidylethanolamine, trehalose monomycolate, TDMand mycocide B were assayed for activity 61,78. Trehalosemono- and dimycolates were found to induce profoundrecruitment of a range of immune effector cells in vivo,indicating that the trehalose mycolates, particularly TDM, are the most bioactive lipids in the mycobacte-rial cell-wall fraction78. The potency of the responseto TDM in comparison to other mycobacterial lipidsis clear even at the histological level. These data areconsistent with the rich body of literature document-ing the bioactivity of trehalose mycolates both in vitro and in vivo51,61,71,72,81–83.

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.

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Matrigel matrix

Lymphocyte cuffing Cells around the beads

Bloodvessels

Fibrouslayer

Polystyrenebead coatedwith TDM

Fat cells at base of peritoneal cavity

Neovascularization

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 functionsThe use of genetics to probe the biological activity of the cell-wall lipids is problematic, owing to the restrictednumber of mutants available because the major classes of lipids seem to be essential. However, three lipid modu-lators have been analysed by mutational approaches,including phthiocerol dimycocerosate (PDIM), TDM

and a phenolic glycolipid. Although the infection studieshave been carried out predominantly in mice, these dataare probably significant to the regulation of pathology atthe infection site in humans.

The activity of TDM in modulating the host inflam-matory response has been confirmed and extended by mutational studies that revealed that TDM is modifiedby an unusual cyclopropane addition. Mutants defectivein either the production of cyclopropane or the additionof cyclopropane to TDM have a markedly enhancedinflammatory response at a comparable bacterial load inmurine infection experiments84–86. This in vivo pathology translated into a five-fold increase in the level of TNF-α

released by infected macrophages in vitro. These resultsare intriguing because they imply that the pathologicalproperties of the lipid have been ‘designed’ to mediate alevel of damage appropriate to the survival and persist-ence strategies of the bacteria.

Signature-tagged mutagenesis screens were used toidentify loci involved in the synthesis and export of PDIM, which resulted in a growth defect in macrophagesand in mice87–89. Interestingly, the growth defect of themutated M. tuberculosis strains in mice seemed to betissue specific, and was restricted to the lung whereasbacterial loads in the liver and spleen were unaffected.The authors hypothesized that the lipid had a role inregulating the immune response within the infected tis-sue site89, although more work is needed to elucidate thetissue-dependency of the phenotype.

Finally, it has long been known that the W-Beijingfamily of human TB isolates are particularly virulent inhumans, and induce extreme pathology at a relatively low bacterial load in mice. Analysis of bacterial gly-colipids in mice and rabbit models identified a phenolic

glycolipid, similar to PDIM, that was unique to theseW-Beijing strains90,91. Deletion of genes in a locus thatencodes the machinery for polyketide and PDIM syn-thesis led to loss of this lipid and a marked reduction inthe hypervirulence phenotype. Analysis of the responseof macrophages to purified phenolic glycolipid indi-cated that the lipid suppressed their proinflammatory response, resulting in depressed production of TNF-α,IL-6, IL-12 and CCL2. How this translates into virulencein vivo remains to be determined but the data recipro-cate the theme that virulence — and pathology — mustbe regulated to yield a pathogen capable of chronicity,reactivation and transmission.

Clearly, a genetic route in generating bacterialmutants with structurally defined defects in theircell-wall constituents is attractive. The data emergingfrom such studies are tantalizing but they underlinethe complexity of the interaction and emphasize thedivide between macrophage experiments in vitro and the results of in vivo challenge in a multicellularenvironment.

TransmissionThis Review has attempted to generate a picture of a strong host immune response that provides thebuilding blocks that form the granulomatous tis-sue 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-wallcomponents of M. tuberculosis can aggravate its hostinto a strong proinflammatory response that causesthe tissue to compartmentalize this persistent irritantbehind a fibrous capsule. Artificial models with iso-lated lipids arrayed on particles reciprocate many of the characteristics of true tubercles during their early stages of development. The real challenge, however, isto understand what the bacterium can do to drive thelate-stage damage that leads to transmission. TDM isan interesting candidate for such a mediator. It seems

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 showsextensive 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.

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Profiling the transcriptional response in defined areasof human TB granulomas through the use of microarray analysis of laser-capture micro-dissected regions fromhuman tissue might provide some insights into the path-ways activated in the host during this crucial transition.The application of this information to more appropriateanimal models such as guinea pigs, rabbits or monkeysmight even allow these pathways to be probed experimen-tally. Although it might be a pipedream to imagine wecould ever block the transmission of M. tuberculosis, weshould at least aim to understand how the propagation of this successful pathogen is achieved.

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AcknowledgementsThis 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 notablyE. Rhoades, R. Geisel and K. Sakamoto.

Competing interests statementThe author declares no competing financial interests.

DATABASESThe 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 INFORMATIONDavid G. Russell’s homepage:http://www.vet.cornell.edu/

public/microbiology/russellnew2.htmAccess to this links box is available online.

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