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Page 1: What's good for the host is good for the bug

What’s good for the host is goodfor the bugJoAnne L. Flynn1 and John Chan2

1Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA2Departments of Medicine and Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

Tuberculosis, caused by Mycobacterium tuberculosis,

kills approximately two million people each year. The

infection is characterized by an inflammatory response

culminating in the formation of a granuloma, a collec-

tion of immune cells that controls the infection.

However, the granuloma can be the source of immuno-

pathology that encourages transmission. Recent data

support the idea that mycobacterial products can

positively and negatively regulate the inflammatory

response. Our contention is that induction of the

immune response and subsequent granuloma for-

mation is beneficial to the host for control of infection,

and is also beneficial to the bacillus, as a place to hide

and as a means for transmitting the infection to naı̈ve

hosts.

Introduction

Mycobacterium tuberculosis is a well-adapted and verysuccessful human pathogen. This organism is believed tohave infected at least one-third of the current world’spopulation. A subset of those people infected progress toprimary tuberculosis, and approximately two millionpeople per year die of this disease. However, mostinfections are controlled by the immune response andare asymptomatic; nonetheless, the bacilli persist in thehost and this is termed latent tuberculosis. It is estimatedthat a latently infected person has a 10% lifetime chance ofreactivating the latent M. tuberculosis infection andprogressing to active tuberculosis. Only individuals withactive tuberculosis are contagious and capable of infectingothers. We believe that M. tuberculosis is dependent oninducing an immune response and subsequent immuno-pathology to provide a niche for survival and also tofacilitate transmission to naı̈ve hosts. Recent datastrongly support a role for mycobacterial products in theregulation of the immune response, suggesting thatinduction of the immune response could be beneficial tothe pathogen, as well as to the host.

Induction of the immune response to M. tuberculosis

The immune response to infection with M. tuberculosisinvolves a strong T-cell response, consisting of CD4 andCD8 T cells that can secrete interferon (IFN)-g to activatemacrophages, in conjunction with another signal, such as

Corresponding author: Flynn, J.L. ([email protected]).

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tumor necrosis factor (TNF; reviewed in Ref. [1]). Thebacterium survives and grows within non-activatedmacrophages, but activated macrophages have variousanti-mycobacterial mechanisms. CD8 T cells can killinfected macrophages and the bacteria within themusing perforin and granulysin [2]. These lymphocytesare primed in the lymph nodes and then migrate to thelung, along with macrophages, and then to the site ofinfection within the lung. This culminates in the for-mation of a granuloma, a collection of immune cells thatfunctions to limit bacterial replication, prevent the spreadof infection, and limit the immunopathological conse-quences of the mycobacterial infection. The tuberculousgranuloma is a contained immune environment for control-lingthe infection,and isusually found inthe lungs,althoughit can be in any organ. Mechanisms regulating granulomaformation are incompletely defined. Evidence suggests thatTNF plays a crucial role in the formation and themaintenance of the tuberculous granuloma in both miceand humans. Paradoxically, TNF also contributes signifi-cantly to the development of immunopathology.

Although the granuloma is well suited for its job oflimiting bacterial replication and dissemination, in manycases the tubercle bacillus adapts to survive within thegranuloma for the lifetime of the host. Therefore, thegranuloma probably serves as a special niche forM. tuberculosis, and the organism possesses severalmechanisms for evading elimination by the immuneresponse (reviewed in Ref. [3]).

The fact that a strong immune response toM. tuberculosis is present in most infected people, andthat most infections are contained for the lifetime of thehost, appears to argue against the success of this pathogenin the human population. It appears to be a widely heldopinion that M. tuberculosis is a master of down-regulat-ing the immune response. We are of the opinion that,although there is certainly immune evasion and modu-lation, there is a strong immune response induced in mostpeople infected with the organism. Instead of preventingan immune response, recent data suggest that certainproperties of M. tuberculosis promote the induction of arobust immune response, leading to the hypothesis thatthe organism benefits from the immunological reaction tothe infection. We are arguing that it is advantageous to thebacillus to promote and modulate T-cell responses andgranuloma formation to ultimately enhance transmissionof the organism to susceptible hosts.

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d. doi:10.1016/j.tim.2005.01.005

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Box 1. The role of TNF in granuloma formation

Infection of a macrophage by Mycobacterium tuberculosis induces

the production of tumor necrosis factor (TNF), as well as other

inflammatory cytokines. TNF acts on macrophages to induce

chemokines, such as CCL5, CCL9, CXCL10 and CCL2, although the

infection also induces a lower level of these chemokines in a TNF-

independentmanner. The chemokines set up a gradient in the tissue,

which is then sensed by immune cells (i.e. T cells and macrophages)

coming into the lungs bearing chemokine receptors (e.g. CCR5,

CXCR3 and CCR2). The chemokines help to guide the cells to the site

of infection, where they interact to form a granuloma; this also

requires TNF to maintain its structure and function. In this case,

chemokines might help to hold cells in place and prevent their

migration away from the infection site. Without TNF, cells enter the

lungs, but do not focus at the site of infection, suggesting a local role

for this cytokine in directing cell migration. This scenario is

supported by the histological findings in the lungs of a patient

with TNF blockade-induced reactivation tuberculosis.

Opinion TRENDS in Microbiology Vol.13 No.3 March 2005 99

Mycobacterial products influence induction of immune

responses

M. tuberculosis interacts initially with alveolar macro-phages and dendritic cells in the airways, and then withtissue and monocyte-derived macrophages as well asdendritic cells in the lungs. The interaction with theseantigen-presenting cells (APCs) results in the productionof inflammatory cytokines, including TNF and interleukin(IL)-12, as well as a variety of chemokines [4,5]. However,each of these cell types are likely to be distinct in theirinteraction with the bacillus, based on differences incombinations of cell-surface molecules, signaling path-ways and sites of action. It has been demonstrated inseveral studies that macrophages and dendritic cellsrespond differently to this infection [4], and this mightbe responsible for differential modulation of the immuneresponse throughout the course of infection.

The initial inflammatory response induced by thebacterium is crucial to the formation of the granuloma.There has been increased interest in the mycobacterialproducts that influence the inflammatory response.Although only a small number of bacterial proteins orlipids that induce these responses have been described todate, it is clear that M. tuberculosis uses several pathwaysto influence the inflammatory response. This suggeststhat the initial inflammatory response, at least at acertain level, is not detrimental to the success ofM. tuberculosis as a pathogen.

The dendritic cell is the primary cell that primes naı̈veTcells to become effector cells, and is very specialized in itsresponse to pathogens and other foreign stimuli. To inducea type 1 (IFN-g producing) T-cell response, IL-12 pro-duction by the dendritic cell is crucial. M. tuberculosisinfection of dendritic cells induces IL-12 production, aswell as other cytokines [4,6]. A heat-shock protein (HSP)produced by M. tuberculosis, known as HSP-70, binds toCD40, an important co-stimulatory molecule on dendriticcells and macrophages, and induces IL-12, as well asseveral chemokines [7–9]. It has been demonstrated, usinga murine tuberculosis model, that this interactionbetween M. tuberculosis and CD40, and the subsequentproduction of IL-12, is essential during a low-dose infectionto induce a protective type 1 T-cell response [9]. This T-cellresponse is crucial for granuloma formation. The 19 kDaand 38 kDa (and other) lipoproteins of mycobacteria havealso been reported to induce IL-12 via a toll-like receptor(TLR)-2 pathway in human cells [10,11].

Lipomannan (LM), a cell-wall lipoglycan, also inducesIL-12 and TNF production by macrophages in a TLR2-dependent manner [12,13]. Interestingly, LM is alsocapable of inhibiting pro-inflammatory cytokines in aTLR-2 independent fashion [12]. Lipoarabinomannan(LAM) does not signal through TLR-2 and does not inducepro-inflammatory cytokines. However, lipoarabinonman-nan (LAM) appears to induce IL-10 through binding to adifferent molecule on dendritic cells, known as DC-SIGN(dendritic cell-specific ICAM-3 grabbing non-integrin)[14]. M. tuberculosis infection can also induce IL-10 andIL-6 production by dendritic cells via a TLR-2-dependentmechanism [15], suggesting differential modulation of thedendritic cell function.

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A combination of factors (e.g. ratio of LM to LAM [16])can lead to an activated dendritic cell, followed by down-regulation of the inflammatory signals once priming of animmune response has occurred. This differential regu-lation is probably dependent on the amount of bacteriawithin or in contact with dendritic cells, either in thelymph nodes (during priming, where bacterial load isprobably low) or in the lungs (within a granuloma, wherebacterial load might be higher). The T-cell response isprimed in the lung-draining lymph nodes of the host, andthen the T cells migrate to the lungs. Without induction ofthe T-cell response, M. tuberculosis infection proceedsunchecked. Thus, this initial dendritic cell–M. tuberculosis interaction is crucial for controlling theinfection and setting up the immune response.

Regulation of granuloma formation

TNF is a key cytokine for granuloma formation, and inmice that lack TNF or the TNF receptor, granulomaformation is aberrant or delayed, and M. tuberculosisinfection is rapidly fatal [17,18]. In mice with chronicinfection, neutralization of TNF results in loss of granu-loma organization, aberrant pathology and subsequentdeath [19]. The significance of this cytokine in humans hasonly been revealed recently. It has been reported thattreatment with TNF blockade using neutralizing anti-bodies in patients with inflammatory disorders, such asrheumatoid arthritis and Crohn’s disease, results inenhanced susceptibility to M. tuberculosis [20]. Epidemio-logical data and the demographics of the study cohortsuggest that the infection associated with TNF blockaderepresent reactivation tuberculosis. Histological lungsamples from a patient with TNF blockade-inducedtuberculosis revealed extensive lymphocytic infiltrationand disorganization of the tuberculous granuloma. Theseobservations support the theory that TNF plays animportant role in the containment of latent tuberculosisand in the maintenance of the structure of the tuberculousgranuloma. Studies of the effect of anti-TNF antibodies inrheumatoid arthritis patients have provided evidence forreduced migration of cells to the joint, suggesting thatTNF might function to control infiltration of cells toinflammatory sites [21] (Box 1).

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Table 1. Mycobacterial proteins and lipids that modulate host

inflammatory responses

Mycobacterial

products

Host factors

modulated

Receptors Refs

19kDa and 38kDa

lipoproteins

IL-12 TLR-2 [10–11]

HSP-70 IL-12 CD40 [7–9]

Lipomannan IL-12, TNF TLR-2C

unknown

[12,13]

Lipoarabinomannan IL-10 (IL-6?) DC-SIGN? [14]

Trehalose

dimycolates

TNF, IL-6, IL-1b Unknown (not

TLR2 or TLR4)

[26–28]

Phenolicglycolipids TNF, IL-6, CCL2 Unknown [30]

ESAT-6/CFP-10 family TNF Unknown [32]

Opinion TRENDS in Microbiology Vol.13 No.3 March 2005100

Chemokines, which provide the signals that lead cellsto the site of infection, are induced by M. tuberculosisinfection, and part of this induction is dependent on TNFexpression by the infected macrophages [22–24]. Neutral-ization of TNF in vitro or in vivo downregulates chemo-kine expression by macrophages at the level of thegranuloma, and prevents adequate granuloma formationand maintenance [24]. There are several mycobacterialmolecules that appear to induce TNF and possiblychemokines, providing a link between the organism andthe induction of a granulomatous response.

The mycobacterial cell wall is lipid-rich, and theselipids can traffic through the endocytic pathway of infectedcells and exit the cell as vesicles that have the ability tointeract with neighboring cells [25]. The trehalose dimy-colate (TDM) of mycobacteria has long been known toinduce a robust granulomatous inflammatory response inthe host [26,27]. Recently, using a mouse model with lipid-coated beads, Rhoades et al. [28] have demonstrated thatvarious mycobacterial lipids cause cells to migrate towardthe bead and form a rudimentary granuloma, without anylive bacteria present. In particular, TDM induced IL-1b,IL-6 and TNF in vivo and in vitro, in a TLR2- and TLR4-independent fashion (D. Russell and E. Rhoades, personalcommunication).

Glickman et al. [29] have recently observed thatdisruption of pcaA, which encodes cyclopropane synthe-tase, an enzyme involved in mycolic acids synthesis,results in attenuation of virulence of M. tuberculosis.The DpcaA M. tuberculosis mutant produced chemicallyaberrant mycolic acids (a component of TDM) as a result ofthe loss of function of the cyclopropane synthetase. Morerecently, Glickman and Porcelli have shown that thealtered DpcaA TDM exhibits diminished ability to inducemacrophage TNF production (M.S. Glickman, personalcommunication). Further, injection of purified DpcaATDM into mice induces remarkably less pulmonic inflam-mation compared with wild-type glycolipid (M.S. Glick-man, personal communication). These data stronglysupport that mycobacterial lipids play a crucial role inthe induction of signals (including TNF) that are import-ant for recruiting cells to the site of infection and thesubsequent formation of a granuloma.

Recently, the production of phenolicglycolipids (PGLs)has been linked to the hypervirulence of clinicalM. tuberculosis isolates. Disruption of pks (polyketidesynthase) 1–15 gene of HN878, a hypervirulent clinicalisolate, leads to PGL deficiency and attenuation ofvirulence [30]. In vitro, macrophages treated with PGLwere impaired in production of inflammatory cytokines,such as TNF and IL-6. Wild-type HN878 induces lesscytokine production from macrophages than the pksknockout, further supporting a role for PGL in down-regulating the host inflammatory response inM. tuberculosis infection. This study linksM. tuberculosis virulence to an anti-inflammatory lipid.

Together, the above results provide strong evidencethat M. tuberculosis lipids, which could be pro- or anti-inflammatory, play an important role in regulating thetuberculous granulomatous response (Table 1). In supportof this notion, it has been reported that Ja281K/K mice,

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which are deficient in the CD1d-restricted natural killerT (NKT) cells, fail to form granulomatous-like lesionssubcutaneously when injected with deproteinized cell wallderived from M. tuberculosis H37Rv [31]. In addition,recruitment of NKT cells to the site of granulomaformation is mediated by mycobacterial glycolipids,particularly phosphatidylinositolmannosides.

These observations do not preclude a role for non-lipidmycobacterial components in modulation of the hostinflammatory response to the tubercle bacillus (Table 1).An M. tuberculosis mutant with disruption of the snm4gene, which encodes a component of a Sec-independentsecretory system, is attenuated for virulence and exhibitsenhanced capacity to stimulate TNF production bymacrophages [32]. The Snm secretion pathway mediatesthe transport of two M. tuberculosis proteins, ESAT-6 andCFP-10, the deficiency of which correlates with virulenceattenuation. Therefore, the Snm, ESAT-6, CFP-10 systemmight represent the protein counterpart of the mycobac-terial phenolicglycolipids in the down-regulation of thehost immune response to the tubercle bacillus. However,both CFP-10 and ESAT-6 are immunodominant proteins ofM. tuberculosis, demonstrating that the host’s ability tomount a response against these proteins is notcompromised.

Clearly, there exist multiple pathways by whichM. tuberculosis can up- or down-regulate the inductionof T-cell responses and the granulomatous response of thehost. The anti- or pro-inflammatory effects of thesepathways are probably determined by the interactionbetween distinct mycobacterial components and specifichost cells, the response of which is in turn dependent onthe genetic make-up of the host. The complexity of thisinteraction is further increased by the probability that theM. tuberculosis factors that can regulate the granuloma-tous response are differentially expressed during thedifferent stages of life cycle in the infected host (Figure 1).For example, it is possible that expression of cyclopropanesynthetase, crucial for the full manifestation of the pro-inflammatory property of TDM by virtue of its ability totrigger TNF production, is at its highest level during theprocess of cavity formation. Therefore, the granulomatousresponse at any given time during infection is the net resultof the interaction between host cells with multiple inflam-mation-regulating M. tuberculosis factors, the expression ofwhich are dependent on the phase of infection. Althoughthese features are technically difficult to address because

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+++ ++++ + +++++

Initialcontact Early Persistence

Reactivation/tissue damage

Phase ofinfection

RelativeTNF

activity

Relativeexpression ofTNF-inducing

M. tuberculosisgenes

Figure 1. Regulation of the granulomatous inflammatory response byMycobacterium tuberculosis: a hypothetical scenario. The relative levels of expression of the four TNF-

inducing M. tuberculosis factors, depicted by the red, green, plum and purple arrows (the length of the arrows correlates positively with the levels of expression), vary with

the different phases of tuberculous infection. Assuming that these four M. tuberculosis components act additively to induce macrophage TNF production, the resultant

relative TNF activity in the tuberculous granuloma of the lungs at a specific phase of infection is determined by the levels of expression of the four factors during that phase of

infection. During the initial interaction betweenM. tuberculosis and macrophages, the four TNF-inducing mycobacterial components result in macrophage TNF production.

As the infection enters the early phase, the expression of the four TNF-inducingM. tuberculosis factors changes in such a way that leads to a net increase in TNF activity in the

granulomatous lesions. This enhanced TNF activity plays a significant role in the rigorous recruitment of immune cells (brown and pink circles and ovals) [24]. As the host

controls the infection, the number of bacilli (depicted as red rods within the macrophage) in the granulomatous lesion decreases. With transition of the infection into the

persistence phase, the expression levels of the TNF-inducingM. tuberculosis components again changes, resulting in a net decrease in granulomatous TNF activity. During

disease recrudescence in the particular host depicted, expression of the TNF-inducing products by the reactivating bacilli results in a robust TNF activity that leads to tissue

damage. Clearly, the granulomatous response is regulated bymultipleM. tuberculosis as well as host factors that in turn, can be differentially expressed at the various phases

of tuberculous infection. In addition, a similar scenario, albeit with potentially different ligand–receptor interactions, can be imagined for dendritic cells during priming of the

response as well as during the chronic phase of infection.

Opinion TRENDS in Microbiology Vol.13 No.3 March 2005 101

modeling stages of tuberculosis can be very challenging,understanding these complex interactions will illuminatethe mechanisms by which granuloma formation is regulatedand M. tuberculosis infection controlled.

Pathology and transmission: the key to successful

pathogenesis

Pathology induced by a microbe is often associatedwith transmission to a new host. In the case oftuberculosis, transmission depends on adequate num-bers of bacteria in the airways aerosolized by thecough or breathing of a person with active tubercu-losis, and it stands to reason that those that cough outlarger numbers of bacteria will be more contagious. Infact, numerous epidemiological and clinical data sup-port a strong association between smear-positivetuberculosis patients (those with enough bacteria insputum to detect by acid-fast staining) and increasedrisk of transmission [33]. Cavitary disease, where agranulomatous necrotic lesion has enlarged and erodedinto the bronchus, spilling millions of bacteria into theairways, is strongly associated with increased trans-mission [34,35].

Children rarely have cavitary tuberculosis and are notcommonly smear-positive; consequently, children are notconsidered to be very contagious [36], although exceptionsexist [37]. AIDS patients also have cavities less frequently,and are more likely to be smear-negative (fewer bacteria

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in sputum) [38,39]. Although it has been believed thatsuch differences in pathology are the result of primaryversus reactivation tuberculosis, more recent studiessupport the theory that these differences are related toimmune status of the host rather than timing ofinfection [39,40]. These findings suggest that effectivetransmission can be associated with granuloma for-mation and eventual breakdown, and support thehypothesis that without an adequate immune responsefor development of a granuloma, the transmission ofbacteria to a susceptible host is greatly reduced.

Although in most cases this immune response willcontain the infection successfully and no transmission willoccur, apparently only a small percentage of infectionsmust become active to continue spread of this infection. Itis estimated that between 2 and 16 new infections can becaused by one case of active TB [41,42], and a major riskfactor for transmission is the presence of a cavity in thelungs [34,35]. Thus, it is to the advantage of the tuberclebacillus to induce a strong immune response, resulting ingranuloma formation and subsequent pathology thatpromotes transmission. It is clear that the organism hasmultiple mechanisms that contribute to and regulateinduction of the immune response as well as granulomaformation and maintenance. An understanding of theseprocesses, with an open mind about what contributes to‘virulence’, will be crucial to the fight against tuberculosis(Box 2).

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Box 2. Outstanding questions

† What is the balance of stimulatory and inhibitory mechanisms

induced by the bacteria?

† Are microbial products that induce strong immune responses

present early, whereas those that inhibit responses expressed later

in infection?

† What are the receptors for these mycobacterial lipids and

proteins?

† What is the effect of the loss of one or more of these components

in the human system?

† Where strains exist in nature that have immunostimulatory or

inhibitory properties, can a link to more extensive disease or

transmission be found?

† What are the mechanisms that mediate liquefaction and cavita-

tion in the granuloma?

Opinion TRENDS in Microbiology Vol.13 No.3 March 2005102

AcknowledgementsWe are indebted to Peter Small and Kathy DeRiemer for helpfuldiscussion. We are grateful to David Russell, Elizabeth Rhoades, MichaelGlickman, Steven Porcelli and Clifton Barry for providing data andinformation before publication. JoAnne L. Flynn is supported by the NIH(AI37859, AI47485, AI50732, HL71241, HL68526 and HL75845) and theAmerican Lung Association (CI-016). John Chan is supported by the NIH(AI50732, HL71241 and HL68526).

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