Leishmaniasis (Nature)

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Leishmaniasis is one of the most significant of theneglected tropical diseases, with 350 million people in 88countries worldwide living at risk of developing one ofthe many forms of the disease1. It is caused by infectionwith one of several different species of protozoan para-sites of the genus Leishmania, which maintain their lifecycle through transmission between an insect (sandfly)and a mammalian host. The flagellated, motile formsof Leishmaniaspp. are called promastigotes. They arefound within the sandfly and progress through variousmorphologically distinct stages of differentiation to ulti-mately become the non-dividing, infectious metacyclicpromastigotes that are transmitted during a sandfly bite.Amastigotes do not have an exteriorized flagellum andlive as intracellular parasites in a variety of mammaliancells, most notably within professional phagocytes suchas macrophages (FIG. 1). Some Leishmaniaspp. causechronic, slow-to-heal diseases that are known as cutane-ous, mucocutaneous or diffuse cutaneous leishmaniasis.In these diseases, the symptoms remain localized to the

skin or mucosal surfaces. Other Leishmaniaspp. dissemi-nate to internal organs such as the liver, spleen and bonemarrow to cause visceral leishmaniasis, which accountsfor most of the ~70,000 deaths per year that are dueto leishmaniasis1. Importantly, the clinical presentation ofleishmaniasis is dependent upon both the parasite speciesand the hosts immune response. For example, Leishmaniamajor, Leishmania mexicana, Leishmania amazonensisandLeishmania braziliensisprimarily cause cutaneous lesions,whereas Leishmania donovaniand Leishmania infantum(known as Leishmania chagasiin South America) cause

visceral leishmaniasis (TABLE 1). Similarly to other intra-cellular pathogens such asMycobacterium tuberculosis or

Mycobacterium leprae, the same microorganism can causea range of diseases depending upon the hosts immuneresponse, including subclinical infections, self-resolvinglesions and chronic disseminated disease.

Recent years have seen major advances in our under-standing of leishmanial biology: the genomes of severalLeishmania spp. have been sequenced2; post-genomicanalysis of these parasites and the host response is pro-ceeding rapidly3; sexual recombination between para-sites has been shown to occur4; and the intricacies of lifewithin an insect vector5,6and of natural transmission7arebeginning to be understood. In this Review, we focus onsome of the major advances from the past few years inour understanding of the immunology and cell biologyof the interactions between the host and Leishmania spp.,and highlight some of the differences in immune regu-lation that have been uncovered from studies of vari-ous Leishmaniaspp. in animal models and in humans.Although this Review is not completely comprehensive,we aim to provide a picture of the complexity that under-

pins the host response to these parasites, and hence tohighlight the potential pitfalls that are associated withdeveloping one simple model for leishmaniasis pathogen-esis. To what extent this complexity derives from intrinsicdifferences in the parasites themselves, from differences inhuman versus mouse host cell responses or from subtle-ties of experimental design remains largely unknown,as few truly comparative studies have been performed.Nevertheless, understanding diversity in hostparasiteinteractions in models, and ultimately in humans, shouldhelp to determine whether new tools for leishmaniasiscontrol can be truly broad spectrum or whether they willrequire specific tailoring to each form of the disease.

*Centre for Immunology and

Infection, Department ofBiology and Hull York

Medical School, University

of York, Wentworth Way,

York YO10 5YW, UK.Penn Institute for

Immunology, Department of

Pathobiology, School of

Veterinary Medicine,

University of Pennsylvania,

Philadelphia, Pennsylvania

19104-4539, USA.

Correspondence to P.K.

e-mail:[email protected]

doi:10.1038/nrmicro2608

Published online 11 July 2011

Leishmaniasis: complexity at thehostpathogen interfacePaul Kaye* and Phillip Scott

Abstract | Leishmaniais a genus of protozoan parasites that are transmitted by the bite of

phlebotomine sandflies and give rise to a range of diseases (collectively known as

leishmaniases) that affect over 150 million people worldwide. Cellular immune mechanisms

have a major role in the control of infections with all Leishmaniaspp. However, as discussed in

this Review, recent evidence suggests that each hostpathogen combination evokesdifferent solutions to the problems of parasite establishment, survival and persistence.

Understanding the extent of this diversity will be increasingly important in ensuring the

development of broadly applicable vaccines, drugs and immunotherapeutic interventions.

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2011 Macmillan Publishers Limited. All rights reserved
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Uptake

Attachment

Phagocyte

Phagolysosome

Intracellular amastigote

Proliferation

Lysis

Reinvasion

Sandfly bite Sandfly bite

Amastigotes

Procyclicpromastigotes

Proliferation in the midgut

Metacyclicpromastigotes

Two-photon intravitalimaging

A fluorescent-laser-based

microscopy technique that

allows real-time imaging of cells

that are deep within the tissues

of live animals.

Chemokines

A family of small (~810 kDa)

chemotactic cytokines that

regulate cell migration and

function. Different chemokine

families are defined by the

presence of specific motifs

known as C, CC, CXC and CX3C.

Host cells for Leishmaniaparasites

Leishmaniaspp. had been widely regarded as fastidious,obligate intracellular pathogens of macrophages, butrecent studies have confirmed that these parasites have afar greater degree of promiscuity in host cell range than

previously thought. Infections of multiple cell types, bothin vitroand in vivo, have been reported, including forhaematopoietic cells that arise from a common myeloidprecursor (BOX 1;FIG. 2), and for non-haematopoieticcells such as fibroblasts. On a cautionary note, parasitesmay spread between cells during tissue homogenization,posing an additional challenge to in vitrostudies of hostrange8,9. In this section, we describe studies on the relativeroles of different cell types as hosts for Leishmaniaspp.

The importance of the neutrophil.One of the major chal-lenges that is faced by the metacyclic promastigote afterit enters the mammalian host is to establish intracellular

residence in long-lived macrophages without trigger-ing their innate antimicrobial defences. In 2003, Laskayand colleagues suggested that neutrophils could act asTrojan Horses to help promastigotes to achieve thisgoal. Through studying L. major, they observed thatpromastigotes were readily phagocytosed by neutrophilsin vitro, but survived within neutrophil phagosomes. Theinfected neutrophils were induced to undergo apopto-sis and became a phagocytic meal for macrophages thatwere added to the culture. As apoptotic neutrophils arephagocytosed through receptor-mediated pathways thatfail to trigger macrophage defence responses10, their car-goes of promastigotes were thereby efficiently and safelyshuttled into the macrophage phagosome11. Neutrophilsare indeed present in the early lesions that follow L. majorinfection in mice, and they are also detectable by histo-pathology in many forms of human leishmaniasis.Two-photon intravital imaginghas recently provided a strik-ingly visual demonstration of the rapidity with whichneutrophils descend upon L. majorpromastigotes aftersandfly (or needle) transmission, and these cells can now

reasonably be regarded as one of the main early hosts forL. majorin vivo12.

Rapid infiltration of neutrophils into the skin is notlimited to those Leishmaniaspp. that cause cutaneousdisease, as it also occurs with L. infantum, which causes

visceral leishmaniasis13. The cues that drive this neutro-phil response are yet to be defined. The rapidity of theresponse and its association with local tissue damage sug-gests a role for alarmins, which are endogenous moleculesthat signal tissue and cell damage14. However, candidatealarmins such as interleukin-33 (IL-33)15have not yetbeen directly measured in leishmaniasis. Mononuclearphagocytes that are infected with Leishmaniaparasitesproduce various chemokines, which are known to attractneutrophils (reviewed in REF. 16). Cytokines also exertcontrol over neutrophil recruitment. For example, IL-17can promote17and type I interferons (IFNs) can inhibit18neutrophil recruitment, but their precise contributionto this immediate immune response is unclear. Finally,unlike needle infection, sandflies may co-transmit bac-teria and viruses, which may help drive inflammatoryresponses19,20. In situ imaging has shown that mostinfected neutrophils, which rapidly engulf promastig-otes on infection, are short-lived and release the parasitesbefore being actively phagocytosed by macrophages12. Infact, the same study showed that the numbers of para-sites in macrophages and dendritic cells (DCs) of mice

are unchanged after neutrophil depletion, which suggeststhat neutrophils may merely scavenge parasites that areotherwise ignored. Further in situ analysis using trans-genic mice to visualize other potential host cells shouldprove to be equally informative (see later). In summary,although neutrophils clearly participate in the earlyresponse to infection, their role as a Trojan Horse has yetto be directly confirmed in vivo.

Although the studies noted above suggest that uptakeby neutrophils contributes to L. majorinfectivity and mayassist in life cycle progression, this may not be the caseduring infections with other Leishmaniaspp., or indeedunder all circumstances with L. major. For example,

Figure 1 |The life cycle of Leishmaniaparasites. Leishmaniaprocyclic promastigotes

differentiate in sandflies into infective, non-dividing metacyclic promastigotes, which

are located ready for transmission at the stomodeal valve (an invagination of the

foregut into the midgut). During blood feeding, the sandfly regurgitates metacyclic

promastigotes, together with immunomodulatory parasite-derived proteophospho-

glycans and various salivary components. The metacyclic promastigotes are then

phagocytosed by one of several possible cell types that are found in the localenvironment (FIG. 2).After establishing an intracellular residence, metacyclic

promastigotes transform into aflagellate amastigotes. Amastigotes undergo

replication within host cells, which rupture when too many amastigotes are present,

allowing reinfection of local phagocytes.The transmission cycle is complete when

infected phagocytes are taken up by another sandfly with the blood meal, and

amastigotes then convert into promastigotes in the sandfly midgut.

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neutrophil depletion in mice increased susceptibility toneedle-inoculated L. braziliensisinfection21. Some earlystudies with L. majorhave suggested that the effects ofneutrophil depletion are mouse strain dependent 22,but these studies often used a monoclonal antibody,RB6-8C5, that is now known to deplete inflammatorymonocytes as well as neutrophils. Contradictory rolesfor neutrophils are also evident from studies of anotherinnate neutrophil function, the extrusion of neutro-phil extracellular traps (NETs), which are composed offilamentous DNA that is decorated with antimicrobialpeptides. In human neutrophils, NET extrusion wasstimulated by L. donovanipromastigotes by a pathwaythat was independent of lipophosphoglycan (LPG)(BOX 2). However, whereas wild-type parasites survivedthis onslaught, LPG-deficient L. donovanidid not23, sug-gesting a parasite-protective role for this major surfaceglycoconjugate. By contrast, wild-type L. amazonensiswas susceptible to killing by NETs, and LPG that wasisolated from these parasites triggered NET release 24.Given the many diverse parts played by neutrophils inthe innate and adaptive immune response (as furtherdiscussed below), it will be technically challengingto directly confirm the importance of parasite transit

through neutrophils and the role of NETs in parasiteestablishment and disease progression in vivo.

Macrophages, monocytes and dendritic cells. Althoughparasites can readily be found in neutrophils, it is withinmononuclear phagocytes that there is the best evidencefor their replication and long-term survival. Two-photonintravital imaging of mouse skin following needle injec-tion of L. majorhas provided direct evidence that der-mal DCs take up parasites within the first few hoursof infection. The process of uptake is highly dynamic,with dermal DCs discriminating between parasites andinert beads and capturing their prey in a process that

involves pseudopodium extension25. Resident dermalmacrophages are also rapidly infected, and they becomethe dominant infected population after 24 hours12.Heterogeneity in the mechanism of phagocytic uptake(for example, coiling versus conventional zipper phago-cytosis) has been reported for different types of macro-phages in vitro26, but the mechanism (or mechanisms)that are used in vivoare not known. It is unclear whetherdermal DCs or macrophages are capable of transportingparasites from the initial site of infection to the lymphnode that drains that site and where acquired immuneresponses are initiated, but both questions are importantfor future study.

As the numbers of resident macrophages and DCsin the skin are too limited to sustain parasite multipli-cation, the progression of infection may require therecruitment of the immediate precursor of these cells,the monocyte. Importantly, this process is regulated byneutrophils27. After cutaneous infection with L. majorin mice, it seems that many of the monocytes that arerecruited to the infection site become monocyte-derived

DCs (moDCs28), which subsequently have apparentlycontradictory roles during the infection. On the onehand, moDCs are permissive host cells for the parasite,thus expanding the developing lesion9. Indeed, they mayfacilitate further monocyte recruitment by increasing theblood supply to the site of infection29. On the other hand,moDCs upregulate expression of major histocompatibil-ity complex class II (MHC class II) molecules, which arecritical for the ability of DCs to act as antigen-presentingcells. These cells also secrete IL-12, which is importantfor the induction of a host-protective T helper 1 (T

H1)-

type response9,28. Indeed, in mice lacking the chemokinereceptor CCR2 (and hence the ability to recruit mono-cytes to the lesions), a non-protective T

H

2 response isinduced by the infection30. Interestingly, whereas moDCsmay be critical for the induction of a T

H1 response, the

non-infected (bystander) DCs could be the key mediatorsof this response. This is supported by the fact that DCsthat are infected in vitrowith L. braziliensispromastigotesdo not upregulate MHC class II or secrete IL-12, whereasuninfected bystander DCs in the same culture do31. Thus,some of the most important functions of DCs in pro-moting protective immunity are inhibited in infectedcells. Bystander activation of DCs (measured by upregu-lation of MHC class II) was also found to occur in thespleen of mice that had been infected with L. donovaniamastigotes, but this study showed no impediment of

function in infected DCs32. Bystander DC activation andconsequently bystander T cell activation may provide ameans for facilitating a rapid T cell response at multiplesystemic sites.

In mouse models of visceral leishmaniasis that useintravenous inoculation of amastigotes to establish vis-ceral infection, a further degree of subtlety in host cellpreference is observed. Amastigotes of L. donovaniarepreferentially taken up by resident tissue macrophages(BOX 1)that lack the CD11b and CD11c markers, suchas marginal zone macrophages and marginal metallo-phils in the spleen, stromal macrophages in the bonemarrow and Kupffer cells in the liver 33. Resident splenic

Table 1 |The main species of Leishmaniathat affect humans*

Main disease manifestation Species

Old World, subgenus Leishmania

Visceral leishmaniasis Leishmania donovaniand Leishmaniainfantum

Cutaneous leishmaniasis Leishmania major, Leishmania tropicaandLeishmania aethiopica

Diffuse cutaneous leishmaniasis L. aethiopica

New World, subgenus Leishmania

Visceral leishmaniasis L. infantum

Cutaneous leishmaniasis L. infantum, Leishmania mexicana, Leishmaniapifanoland Leishmania amazonensis

Diffuse cutaneous leishmaniasis L. mexicanaand L. amazonensis

New World, subgenus Viannia

Cutaneous leishmaniasis Leishmania braziliensis, Leishmaniaguyanensis, Leishmania panamensisandLeishmania peruviana

Mucocutaneous leishmaniasis L. braziliensisand L. panamensis

*Adapted from REF. 119.

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Phagolysosome

Metacyclicpromastigote

Sandfly

Dermis

Tissue-residentmacrophage or DC

Neutrophil

Non-leishmanicidalvacuole

Blood vessel

To the lymph node?

moDC

macrophages that express epidermal growth factor-likemodule-containing mucin-like hormone receptor-like 1(EMR1) and are experimentally defined by the mono-clonal antibody F4/80, and resident DCs in spleen andliver are infected to a far lesser extent. Furthermore, incontrast to the dominance of moDCs in both early andlate stages of cutaneous leishmaniasis, moDCs appear tohave a lesser role as hosts for L. donovani8. However, intissues that contain multiple populations of potential hostcells, apparent host cell preference may reflect both dif-fering levels of host cell permissiveness to infection andanatomical constraints to infection.

Stromal cells as host cells for Leishmaniaparasites.Thepresence of L. majorin skin and lymph node fibroblasts

was first highlighted over a decade ago34, and since thattime it has become apparent that infection of stromalcells can also occur in other forms of leishmaniasis. Itwas initially proposed that the residence of L. majorinthese cells provided a niche for survival in the face ofimmune attack, but recent studies also suggest that theinfection of stromal cells can contribute to immune eva-sion. For example, splenic stromal cells that are infectedwith L. donovaniexpress high levels of the chemokineCCL8. Together with CXCL12, this chemokine attractshaematopoietic stem cells into a niche that favours devel-opment of DCs that can reduce the effectiveness of T

H1

immunity35.

The intracellular fate of Leishmaniaparasites

The realization that Leishmaniaparasites can survivein diverse populations of phagocytes, which containphagosomes with differing microbicidal properties,raises an important question: do Leishmaniaparasitesrely on constitutive broad-acting countermeasures forprotection against the phagosomal environment, or dothey employ inducible virulence factors that are selec-tively expressed in differing intracellular environments?As yet, there are few answers to this question, probablyfor a number of reasons. First, post-genomic analysis ofleishmanial virulence factors is still in its infancy. Second,although it is the amastigote that ultimately inhabits andreplicates within the phagolysosomal niche, most studieson intracellular survival have focused on the early estab-

lishment of promastigotes, hence modelling only the firstwave of intracellular infection. Third, the historical useof easily accessible macrophage populations (for example,those derived from peritoneum or bone marrow) ignoresthe tissue-specific heterogeneity of the host cells36.Nevertheless, despite these limitations in studies to date,the complexity to come is already becoming evident.

Phagosome biogenesis.In mouse neutrophils, L. donovanimetacyclic promastigotes survive undamaged in thephagosome an intracellular membrane-bound com-partment that is related to the endoplasmic reticulum(ER)37 providing a mechanistic basis for the Trojan

Figure 2 |Multiple cell types are involved in the uptake of Leishmaniaparasites. Metacyclic promastigotes are

deposited in the dermis in a mixture of immunomodulatory salivary secretions and parasite-derived proteophosphoglycans.

The impact of these factors on local phagocyte function is poorly defined. Metacyclic promastigotes from the initialinoculum (or those that have been released from infected neutrophils) are phagocytosed by tissue-resident macrophages

and dermal dendritic cells (DCs). Capillary and other tissue damage resulting from the mechanical trauma of the bite may

result in the release of endothelial alarmins, such as interleukin-33 (IL-33), which facilitates the recruitment of neutrophils. The

neutrophils swarm around the extracellular metacyclic promastigotes, engulfing many in non-leishmanicidal vacuoles.

The death of neutrophils releases metacyclic promastigotes that may be pre-conditioned for survival in other myeloid

cells. Alarmins (such as high mobility group protein B1 (HMGB1) and IL-1), which are released from ruptured neutrophils,possibly aid in attracting inflammatory monocyte-derived DCs (moDCs) to the local site. Infected inflammatory moDCs

may facilitate parasite traffic to the draining lymph node. Long-term replication and perpetuation of the pathogen

principally involves either macrophages or moDCs, depending on the parasite species. It is not known whether neutrophils

are involved in amastigote uptake after the initial infection.

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Granules

Cytosolic particles within

neutrophils that are referred to

as primary, tertiary or specific,

based on their staining

characteristics and their

content. Primary granules (also

called azurophilic granules)

contain the enzyme

myeloperoxidase, which is

responsible for the green

colour of pus.

RAB GTPases

A large family of monomericguanine nucleotide binding

proteins that act as important

regulators of vesicle transport

and docking in eukaryotic cells.

Fc receptors

Cell surface receptors that

recognize the Fc tails of

antibody molecules. The

binding of particles that are

coated with antibody by the

Fc receptors on phagocytic

cells triggers internalisation

and potent antimicrobial

effector responses.

Horse model referred to earlier. Maintenance of thisER-like compartment requires LPG-dependent inhibi-tion of lysosomal fusion (a function for LPG that was firstdescribed when studying the infection of macrophages38).Thus, LPG acts as a virulence factor to allow safe pas-sage of metacyclic promastigotes through neutrophils,in addition to its other numerous roles. However, a ratherdifferent picture has emerged from studies of humanneutrophils that were infected with L. majoror L. donovani,in which no evidence for the involvement of the ER inphagosome formation was found39. In these neutrophils,primary granules could fuse with parasite-containingphagosomes, but parasite survival was ultimately deter-mined by whether or not fusion of the phagosome withtertiary and specific granules (which are responsible for

acidification and superoxide generation, respectively)had occurred (FIG. 3a).

In macrophages, it is generally accepted that parasite-containing phagosomes undergo maturation to acquirelysosomal properties, and promastigotes inhibit thisprocess. However, early events in phagosome biogen-esis are less clear. In support of an ER-mediated modelof phagocytosis, one study found that most phagosomesthat are formed around L. donovaniand Leishmaniapifanoipromastigotes in bone marrow-derived macro-phages contain the ER chaperone calnexin as well asSEC22b (a vesicle protein that is involved in ERGolgitransport). Using the toxin ricin as a tool to monitor

ERphagosome communication, this study further sug-gested that the ER and the phagosome were in continu-ous communication40. Delayed phagosomal maturation,first demonstrated with L. donovani 38, has also beenobserved using bone marrow-derived macrophages thatwere infected withL. mexicana. In this elegant study,many of the technical challenges of analysing asynchro-nous processes such as phagocytosis were overcome byusing real-time imaging of infection in macrophages thatwere derived from transgenic mice expressing RAB5(which is one of several RAB GTPasesand a key orches-trator of endosome and phagosome maturation) fused toGFP41. Surprisingly, RAB5 was detected in L. mexicana-containing phagosomes for only 12 min, indicating therapid maturation of these organelles. Phagosome matu-ration was delayed (measured by an increase in the timethat the parasite spent in a RAB5+compartment) whenLPG1-deficient promastigotes (which do not undergocomplement receptor-mediated phagocytosis) were used,or when wild-type parasites were opsonized with anti-body to target them for uptake by Fc receptors. However,

in contrast to earlier reports38, LPG-dependent inhibi-tion of phagosome maturation in this model did notultimately affect parasite intracellular survival41.

Various studies have demonstrated that the acidifi-cation of phagosomes that typically occurs during theirtransformation into phagolysosomes is transiently inhib-ited in parasite-containing phagosomes. For L. donovani,this has been reported to be due to the integration of LPGinto lipid microdomainsof the phagosome membrane thatcontain the ganglioside GM1. This process leads to exclu-sion or loss of synaptotagmin V, which is an essentialplayer in the recruitment of the vesicular proton ATPase(V-ATPase) that drives phagosome acidification42. Hence,LPG-deficient parasites were assumed to die as a result ofthe acidification that occurred before they became fullyadapted to an intracellular lifestyle. However, the obser-

vation that LPG-deficientL. majoris also rapidly killed inmacrophages that lack signal transducer and activator oftranscription 1 (STAT1), in which phagosome acidifica-tion fails owing to defective chloride channel function,suggests that the death of LPG-deficient L. majoris inde-pendent of acidification. Indeed, in these latter studies, ahost-protective effect for STAT1 dependent phagosomeacidification was observed to operate only on amastig-otes43. Related to this observation, a recent study has sug-gested that the shift in temperature that is associated withthe move from an invertebrate vector to a mammalian

host is the major determinant of amastigote-specific geneexpression44, rather than phagosome acidification.

In addition to the parasite- and cell type-specific dif-ferences noted above, host cell maturation status canalso influence the intracellular fate of Leishmaniapara-sites. For example, the maturation of parasite-containingphagosomes in immature bone marrow DCs is arrestedat the stage of the late endosome, whereas in maturebone marrow DCs the parasite-containing phagosomesacquire the small GTPase RAB7 and fuse with lysosomes(FIG. 3b). This might provide a mechanism for ensuringthat the immature DCs that are infected at local sites cantransport live parasites to systemic tissue. Unfortunately,

Box 1 | Monocyte and macrophage diversity

Monocytes, as well as neutrophils, originate in the bone marrow from a common

myeloid precursor cell. Monocytes have a critical role in leishmanial infections as they

differentiate into macrophages, which provide a home for parasite replication, but

can also differentiate into dendritic cells (DCs), which are crucial in priming a

protective immune response. The macrophages and DCs that arise from monocytes

exhibit many different phenotypes, which are thought to be determined by cues

within the infected tissues114

. In addition, monocytes are a heterogeneous populationof cells, which may also contribute to the generation of distinct macrophage and DC

subsets115. In humans, the best described subsets are classic monocytes, which are

defined by their expression of CD14, and pro-inflammatory monocytes, which in

addition to CD14 also express the low-affinity Fc receptor CD16. Different markers

define monocyte subsets in mice. One monocyte subset expresses CCR2 (a

chemokine receptor that is critical for release of cells from the bone marrow) and

LY6C (the function of which is unknown) and is recruited into inflammatory sites.

Another subset is potentially derived from LY6Chimonocytes and expresses the

fractalkine receptor, CX3CR1. The LY6Chimonocytes that migrate into leishmanial

lesions are the source of monocyte-derived DCs. Our understanding of how these

diverse monocyte subsets contribute to disease is still limited.

Macrophages also display considerable tissue-specific heterogeneity, reflected both

in function and phenotype. For example, in the spleen there are distinct populations

of macrophages in the red pulp (where they express F4/80) and the marginal zone

(where macrophages at the outer border express SIGNR1 (also known as CD209antigen-like protein B) and macrophages at the inner border, which are known as

metallophilic macrophages, express CD169). In lymph nodes, macrophages in the

subcapsular sinus have a defined role in antigen capture. In liver, resident tissue

macrophages are called Kupffer cells, but even these may be heterogeneous. Resident

tissue macrophages may have conventional roles for example, as phagocytes but

some may also have a supportive role in maintaining tissue structure or forming part of

the stem cell niche. As some of these macrophages have functions that are reminiscent

of fibroblasts, they may be referred to as stromal macrophages. Our understanding of

how these different types of macrophages contribute to the various forms of

leishmaniasis is very rudimentary8,33,72.

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Lipid microdomains

Regions of a biological

membrane that are enriched

in a particular protein or

lipid and can vary in size,

composition and function.

For example, lipid rafts are

highly dynamic microdomains,

10200 nm in size, that are

enriched in cholesterol and

sphingolipids and serve to

compartmentalize different

membrane functions.

similar studies examining phagosome biology inother host cells under various conditions of activationare lacking.

In infections with some Leishmania parasites fromthe New World, the parasite-containing phagosomes arestrikingly enlarged, and it has recently been suggestedthat this may help dilute the leishmanicidal effects ofnitric oxide. Lysosome size in cells is regulated by lyso-somal trafficking regulator (LYST; also known as Beigeprotein). Mice with mutations in Lyst and humans withChdiakHigashi syndrome (who carry mutations inthe human homologue, LYST) have large lysosomes andlysosome-related organelles, including phagosomes.When LYST was overexpressed in mouse macrophagesand fibroblasts, phagosomes were reduced in size and theparasites contained within them were more susceptible tokilling by nitric oxide. As LYST is induced during infec-tion with Leishmania parasites, it therefore behaves as aninducible innate response gene45.

Iron regulation in the phagosome.Although manyfactors contribute to the survival of Leishmaniapara-sites in the phagosome, access to iron has a central role(reviewed in REF. 46)(FIG. 4). Amastigotes can use ferrous

iron (Fe2+) as well as hemin (the Fe3+oxidation productof haem) and haemoglobin, and a high-affinity heminreceptor has recently been described in L. infantum47. Inmice, Slc11a1(also known as Nramp1 andLsh) encodesa phagosomal efflux pump that translocates Fe2+andMn2+into the cytosol48and thus limits iron availabilityto the parasite. As a countermeasure, L. amazonensisupregulates the expression of its own iron transporter,LIT1, after its entry into macrophages46,49.Recent stud-ies with L. donovani suggest that intra-phagosomalcompetition for iron may, by depleting the macrophagelabile-iron pool, activate the cytosolic iron sensorsIRP1 and IRP2, which leads indirectly to increased

production of the iron-binding protein transferrin andthus transferrin-mediated iron uptake50.

Subverting intracellular signalling

As Leishmaniaparasites inhabit an intracellular niche,it is perhaps not surprising that they have evolved vari-ous means to attenuate and/or subvert how their host cellintegrates signals from the external immune environ-ment. Indeed the literature has a rich history of reports ofLeishmaniaspp. affecting a range of diverse macrophagefunctions, including chemotaxis, cytokine productionand immune synapse formation. However, until recentlymany of these studies have been overshadowed at amechanistic level by the vexed issue of spatial segrega-tionthat is, how do intra-phagosomal parasites affectthe cytosolic signalling pathways of their host cell? In thissection we review recent studies that are beginning toprovide some clarity to this issue.

Cellular phosphotyrosine phosphatases.The role of a hostprotein phosphatase, SRC homology 2 domain phospho-

tyrosine phosphatase 1 (SHP1; also known as PTPN6), inregulating anti-leishmanial immunity has long been rec-ognized (reviewed in REF. 51). SHP1 has a central role inthe regulation of inducible nitric oxide synthase (iNOS)and hence nitric oxide production, and it was recentlyshown that SHP1 binds to a conserved KTIM motif thatis found in multiple kinases that act downstream of SHP1and are associated with innate immunity (these kinasesinclude extracellular signal-related kinase 1 (ERK1),ERK2 and IL-1 receptor-associated kinase 1 (IRAK1)).L. donovaniincreases SHP1 activity and expression inhost macrophages, a process thought to contribute toparasite survival. But how is this achieved? New evidencepoints to a well-known leishmanial virulence factor,major surface protease (MSP; also known as GP63 andleishmanolysin)5254. MSP enters the host cell cytosol in aprocess that is dependent on the presence of cholesterol-rich lipid microdomains or lipid rafts in the host cellmembrane55, and appears to have an ability to selectivelycleave intra-cytosolic protein tyrosine phosphatases suchas SHP1, protein tyrosine phosphatase 1B (PTP1B) andT cell protein tyrosine phosphatase (TCPTP; also knownas PTPN2), leading to their activation55,56. The kinetics ofprotein tyrosine phosphatase cleavage after exposure toparasites or to recombinant MSP suggests that the raft-dependent entry of MSP into the host cell cytosol mayprecede internalization, but it is unclear whether this

continues while the parasite is within the phagosome.Leishmaniaspp. contain multiple MSP genes, and speciesdifferences in expression of these genes are well known;for example, in L. chagasiMSPs originate from three geneclasses and, in this species at least, there are importantdifferences in glycosylation and cellular localizationbetween MSPs in promastigotes and amastigotes54. It willbe important to determine whether all MSPs cleave intra-cytosolic protein tyrosine phosphatases. In L. donovani,MSP can be found on the promastigote surface as well asin the parasite cytoplasm, and it has been proposed thatthese pools of MSP are functionally distinct, with the sur-face MSP being involved in parasite development within

Box 2 | Surface coat of leishmanial promastigotes

Leishmaniapromastigotes are covered by a dense glycocalyx, which is composed of

several types of molecules that are attached to the plasma membrane using a glyco-

phospoinositol (GPI) anchor. The most abundant molecule is lipophosphoglycan (LPG),

which has an important role in the interactions between the parasite and the sandfly

and, once in the mammalian host, promotes the infectivity of the parasites. LPG is a long

phosphoglycan polymer with multiple repeating sugar residues, glycan side chains and

a capping oligosaccharide. Importantly, there is considerable diversity in LPG structureamong Leishmaniaspp., which explains their vector specificity116and may also affect

their immunological properties. A series of LPG mutants have been developed in

Leishmania spp. that have been extensively used to define the function of LPG both

within the sandfly and within the mammalian host117. Changes in LPG structure as the

parasites transform from a procyclic form to an infective metacyclic form are critical for

releasing the parasites from the sandfly midgut, as well as protecting the promastigotes

in the mammalian host from a variety of immune responses. The surface of

promastigotes also contains a variety of other phosphoglycans and GPI-anchored

glycoproteins. The most abundant surface glycoprotein is the zinc metalloproteinase

MSP(also known as GP63)55, which seems to have a significant role in leishmanial

virulence. Perhaps unsurprisingly, the expression levels of MSP, LPG, glycoinositol

phospholipids and other molecules that are associated with the hostparasite

interaction may vary between Leishmaniaspecies, strain and life cycle stage.

Broad-ranging, comprehensive analysis is therefore required to ascertain the biological

significance of this and other leishmanial virulence factors.

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Phagosome

Promastigote

Phagosome

Lysosome

a bNeutrophil DC

MPO-containingprimary granule

Tertiarygranule

Specificgranule

Parasitedegradation

Parasitedegradation

Parasitesurvival

Parasitesurvival

Immature DCs Mature DCs

LAMP1and LAMP2

CD68

RAB7

Signalosome

A high-molecular-mass

cytosolic or membrane-bound

complex that contains many of

the individual proteins that are

associated with a signalling

pathway, often together with

various scaffold proteins.

the sandfly and the cytoplasmic MSP being a pre-formedstore that is ready for rapid use in the mammalian host57,in a manner analogous to the various effectors that areused during type III secretion in bacteria58.

MSP is not alone in targeting SHP1, as the L. dono-vanipromastigote fructose-1,6-bisphosphate aldolaseimmunoprecipitates with SHP1 from macrophage cyto-sol. Likewise, MSP is somewhat promiscuous in its action;in fibroblasts, transforming growth factor--activatedkinase 1 (an upstream activator of p38 mitogen-activatedprotein kinase (p38 MAPK; also known as MAPK14)) isa target of enzymatic cleavage by MSP, providing a mech-anism for inactivating p38 MAPK56. Similarly, MSP alsoaffects peri-phagosomal actin accumulation, an essen-tial feature of phagosome maturation, by cleaving the

120 kDa scaffold protein tyrosine phosphatase PTPPEST,which is a key regulator of WiskottAldrich syndromeprotein (WASP) and actin remodelling56.

Host cellular protein tyrosine phosphatases may alsohave broader roles in anti-leishmanial immunity. Ofnote, SHP2 (also known as PTPN11), which shares manydownstream targets with SHP1, is coupled to signal-regulatory protein 1 (SIRP1; also known as PTPNS1),which is a myeloid-restricted inhibitory receptor59,60thatbinds the membrane glycoprotein CD47 when macro-phages encounter invariant natural killer T cells duringearly L. donovaniinfection61. Intriguingly, sodium stibo-gluconate, the first-line anti-leishmanial drug in most

parts of the world, also targets SHP1 at concentrationsthat are used for chemotherapy in humans62. Finally,and perhaps not surprisingly, the targeting of cellularphosphatases may not be a ubiquitous virulence mech-anism, and the disparate results for parasite survivaland lesion development that are seen in SHP1-deficientmacrophages and mice may reflect host genotype,the macrophages used (peritoneal versus a cell line), theuse of complement opsonization in vitro and eventhe route of inoculation63,64.

Protein kinase C and nuclear factor-B.Interference withhost cell signalling at the level of macrophage proteinkinase C (PKC) has also long been known65. An enzymewith PKC-like activity that is found in the infective pro-mastigotes of L. mexicanahas been recently reported tobe upregulated during early contact with macrophagesand to be involved in macrophage invasion66. Similarly, arole has been proposed for phosphatidylinositol kinasesin a variety of leishmanial signalling responses that arelinked to invasion. Hyporesponsiveness of host MAPK

and nuclear factor-B (NF-B) leads to cross-toleranceto activation by bacterial lipopolysaccharide (LPS) andL. major67,68but does not appear to be due to the induc-tion of cellular protein tyrosine phosphatases, suggestingalternative means for parasites to inhibit host responsive-ness. L. amazonensispromotes the activity of the NF-Bp50p50 transcriptional-repressor complex, which nega-tively regulates iNOS gene expression. Furthermore, inL. amazonensis-infected macrophages that had been pre-

viously stimulated with LPS, the NF-B p50p50 dimerreplaced the NF-B p65p50 dimer (which is normallyproduced in LPS-stimulated macrophages). In addition,MSP cleaves the NF-B p65 subunit51. These resultssuggest an active process of immune deviation69.

Lipid microdomains.Lipid microdomains, includingrafts and caveolae, have an important role in the organi-zation of membrane proteins, in cellcell contact and innumerous signalling processes. Not surprisingly, they areincreasingly being implicated in the interactions betweenmacrophages and Leishmaniaparasites (FIG. 5). As notedabove, host cell lipid rafts may have a role in the entry ofleishmanial virulence factors such as MSP into the hostcell cytosol. However, rafts and other lipid microdomainsmay also serve as targets for disrupting host cell functionand, indeed, as a portal of parasite entry70. Microarrayanalysis indicates that macrophage lipid metabolic

pathways are perturbed by Leishmania infection71,72.Depletion of cholesterol, which is the major lipid com-ponent of lipid rafts, is observed in a variety of experi-mental and clinical settings and has been associatedwith defective antigen presentation following L. dono-vaniinfection73. Strikingly, this defect can be improvedby cholesterol replenishment, with dramatic effects ondisease progression in vivo. Cholesterol depletion hasalso been implicated in immune modulation by L. major.In L. major-infected macrophages, CD40 is preferen-tially associated with a signalosomecontaining TRAF6(tumour necrosis factor (TNF) receptor-associated fac-tor 6) and spleen tyrosine kinase (SYK), which results

Figure 3 |Phagosome fate is determined by factors from both the host and

Leishmaniaspp. a| In human neutrophils, all phagosomes containing promastigotes

fuse with myeloperoxidase (MPO)-containing primary granules. However, destruction

of the parasites requires the additional fusion of tertiary and specific granules. Fusion of

tertiary and specific granules induces a decrease in luminal pH and also results in an

increase in the concentration of reactive oxygen radicals. In mouse neutrophils, it hasbeen suggested that lipophosphoglycan (LPG) from the parasite has a role in regulating

phagosome fate, but the fusion of specific granules has not been determined. b| Late

endosomal markers such as lysosome-associated membrane protein 1 (LAMP1) and

LAMP2 are found in phagosomes containing Leishmania majorpromastigotes in

both immature dendritic cells (DCs) and mature DCs from mouse bone marrow. By

contrast, recruitment of the small GTPase RAB7, which facilitates lysosomal fusion,

was not observed in immature DCs; this suggests that inhibition of RAB7 recruitment

could be a mechanism that is used by Leishmania spp. to ensure the transport of live

parasites to lymph nodes118.

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Fe3+

TFFe3+

TF

TFR

Fe3+

Fe2+

Fe2+

Macrophagecytoplasm

Amastigote

Phagolysosome

Fe2+

Fe2+

Fe2+

SLC11A1

SLC11A2

TF

LIT1

in ERK1- and/or ERK2-dependent secretion of IL-10.This contrasts with the normal association of CD40 inuninfected cells with a signalosome that contains TRAF2,TRAF3, TRAF5 and the SRC kinase LYN and that leadsto p38 MAPK-dependent IL-12 production74. In addi-tion, L. majorinfection of macrophages has also beenshown to induce MAPK phosphatase 1 (MKP1), whichdephosphorylates p38 MAPK and reciprocally decreasesexpression of MKP3, a phosphatase with specificityfor ERK1 and ERK2 (REF. 75). Thus, parasite-mediatedperturbation of membrane and cytosolic componentsof the CD40 signalling pathway may influence theimmunoregulatory function of macrophages.

Lipid microdomains may also underlie the promiscu-ous functions of LPG. Following L. donovaniinfection,LPG associates with the phagolysosome membrane,regulating the accumulation of periphagosomal fila-mentous actin (F-actin) and thereby transiently inhibit-ing phagosomelysosome fusion (see earlier). A recentstudy indicates that LPG is associated with rafts on thephagolysosome membrane and that, after raft disruption,this and other functions of LPG are lost76. Raft-associated

LPG may also be responsible for the alteration in the sus-ceptibility of L. infantum-infected macrophages to HIVinfection77.

A role for exosomes.Exosomes are vesicles that bud fromthe plasma membrane, and have been found in cell-freesupernatants from a variety of cultured mammalian cellsand pathogens, including Leishmania spp. (reviewedin REF. 78). Recent proteomic analysis of leishmanialexosomes suggests that they contain many if not all ofthe molecules that have functionally been described asleishmanial virulence factors79. Hence, it is possible thatexosomes represent vehicles that allow the entry of these

virulence factors into the host cytoplasm, through fusionwith the mammalian cell plasma membrane or with thephagolysosome membrane. Although exosomes fromL. donovaniand L. majorhave some influence on dis-ease outcome when administered before experimentalinfection80, the precise role of exosomes in pathogenesisremains unclear, as there are no known means throughwhich their production by parasites can be manipulatedin vivo.

Type I interferons.Type I IFN responses are usuallyassociated with viral infections; however, both type IIFNs themselves and the signature genes they induce areincreasingly becoming seen as important in leishmaniasis.One example of such a response being beneficial to theparasite is seen during infection with L. amazonensis,which induces the expression in macrophages of PKR,a protein kinase that is activated by double-strandedRNA. PKR appears to promote parasite survival throughinduction of the macrophage-deactivating cytokineIL-10 (REF. 81). By contrast, there are other examples for

which type I IFN-induced proteins appear detrimen-tal rather than beneficial to intracellular survival. Forexample, interferon regulatory factor 7 (IRF7), which isa master regulator of the type I IFN response, acts as anupstream regulator of leishmanicidal activity in L. dono-vani-infected stromal macrophages 72. But perhaps themost elegant example is that of Leishmaniaguyanensis,which is associated with mucocutaneous leishmaniasisand can carry its own virus (LeishmaniaRNA virus-1;LRV-1)82. The metastatic potential of different L.guy-anensisisolates correlates with the LRV-1 viral load.Importantly, the presence of LRV-1 was associated withincreased Toll-like receptor 3 (TLR3)-dependent secre-tion of IFN and other pro-inflammatory cytokines frommacrophages. Furthermore, cutaneous lesion develop-ment was reduced in Tlr3/mice compared with wild-type mice when both were infected with L.guyanensisstrains that had high levels of LRV-1, but no differencewas observed when both were infected with L.guyanen-sisstrains that had low levels of or lacked LRV-1. Hence,the inflammatory potential of L.guyanensisis dependentupon the virus that it carries83.

Adaptive immunity

Host cells for Leishmania spp.also have a pivotal role inbridging the gap between innate and adaptive immunity.However, although many of the survival strategies noted

above may indirectly affect the three signals that arerequired for inducing T cell activation and differentiation(antigen processing and presentation, the expression ofco-stimulatory molecules and the production of host cellcytokines), there have been surprisingly few studies overthe past few years that directly address these pathways inan in vivocontext and/or in relation to defined virulencefactors or vaccine candidate antigens. Notably, few exam-ples have been found of bona fide virulence factors thatselectively target antigen presentation, although these areabundant in other intracellular pathogens. The tools ofin situ analysis hold great promise for addressing someof these questions in coming years. By contrast, there has

Figure 4 |Iron wars within macrophages infected with Leishmania parasites. Fe2+

transporters from both the host (SLC11A1) and Leishmaniaspp. (LIT1) compete for

phagosomal free iron. Studies in mouse macrophages that were infected with Leishmania

donovanisuggest that the resulting depletion of cytosolic Fe2+may lead to activation of

the host iron-responsive element binding proteins IRP1 and IRP2 (not shown)50. These

proteins enhance the stability of the transferrin (TF) receptor (TFR) mRNA, which in turn

leads to increased TFR-dependent uptake of extracellular Fe3+

. Reduction of Fe3+

to Fe2+

within the early endosome is followed by iron transport into the cytosol, which is

dependent upon the host transporter SLC11A2.

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Phagosome

Promastigote

Virulence factorsCD40MHCII

Lysosome

Plasma membrane

Actinremodelling

Altered antigen presentation

LPG-modifiedlipid microdomain

Lipid microdomain

been, and continues to be, much interest in defining thecellular mediators of acquired resistance. We describein this section some of the more recent nuances of theacquired immune response to Leishmania spp.

Host resistance and adaptive immunity. CD4+TH1 cells

are critical for the control of Leishmaniainfections,owing to their ability to make IFN, which activatesmacrophages and DCs, leading to parasite death (FIG. 6).The role of CD8+T cells in cutaneous leishmaniasis hasbeen less well appreciated. Although early studies indi-cated that CD8+T cells were important to control visceralleishmaniasis84, the initial studies with L. majorindicatedthat CD8+T cells were not important for control of a pri-mary infection but they participated in the resistance toreinfection85. However, when mice were infected with lowdoses of parasites, CD8+T cells appeared to be essen-tial for resolution of primary infection86, owing in partto the ability of IFN to promote a T

H1-type response87.

Nevertheless, CD8+T cells are not always associated

with disease resolution. For example, the recruitment ofCD8+T cells that express the granule-associated serineprotease granzyme B is correlated with lesion progressionin patients infected with L. braziliensis88. The factors thatdetermine when CD8+T cells are protective and whenthey promote disease remain to be elucidated. Moreover,it appears that CD8+T cell exhaustion following infec-tion with L. donovanimay contribute to the chronicityof infection89.

As noted above, moDCs that infiltrate the lesionsappear to be particularly important in the inductionof a CD4+T

H1-type response following L. majorinfec-

tion. Interestingly, it was recently found that CD8+T cell

activation following L. majorinfection appears to insteaddepend upon dermal DCs (defined by expression of theC-type lectin langerin (also known as CD207)). Thus,when dermal DCs were specifically depleted in mice (byadministering diphtheria toxin to mice that expressedthe simian diphtheria toxin receptor under control of thelangerin promoter), CD8+T cell responses were signifi-cantly diminished, but CD4+T cell responses were notaffected90. Importantly, CD8+T cells that are activatedduring Leishmaniainfections may not all be specific, asuninfected DCs that have matured during inflamma-tion can stimulate CD8+T cells to proliferate without theexpression of their cognate ligands32. The role of thesenon-leishmanial-specific CD8+T cells in the infection isnot clear, but when previously activated CD8+T cells areexpanded during infection, they can provide increasedresistance to previously encountered pathogens91.

Parasite persistence.In spite of the development of arobust immune response in resistant mice, as well as inmany patients, a small number of parasites persist fol-

lowing disease resolution. The production of IL-10 has alarge role in dampening the immune response and thusallowing some parasites to escape destruction. This wasshown by depleting IL-10 from mice that were infectedwith low doses of L. major, which led to sterile cure (thatis, no living parasites remained)92. Recent studies haveshown that IL-10 can come from a variety of sourcesfollowing leishmanial infections, including regulatory T(T

Reg)cells93, T

H1 cells9496, CD8+T cells92, B cells97, natural

killer cells98, regulatory DCs35, macrophages99and neutro-phils100. Which of these is most important as a sourceof IL-10 is less clear, but may depend upon differencesin the parasites and the stages of the infection. On onehand, IL-10-transgenic mice (in which IL-10 is only pro-duced in cells that express MHC class II molecules) areextremely susceptible to infection with L. major101. Onthe other hand, a recent study in mice expressing humanIL-10 (from a transgene encoded on a bacterial artificialchromosome) suggested that IL-10 production by T cells,rather than macrophages, may be more critical for para-site clearance102. In L. donovaniinfection, CD8+CD40+T cells may act as contra-T

Regcells by limiting the pro-

duction of IL-10 during the early phase of infection, butthemselves become susceptible to IL-10-induced apop-tosis as the disease progresses103. Thus, the question ofhow immunoregulatory mechanisms limit immuneresponses, and as a consequence lead to the persistence

of parasites following disease resolution, remains anarea of active investigation.

When leishmaniasis involves infection of lymphoidtissues, such as the spleen and lymph nodes, theseundergo dramatic remodelling33,104. Although erosion ofstromal cells and vascular remodelling represent the mostlikely underlying mechanism behind the loss of archi-tectural integrity, the main functional consequence maybe to develop a state of immune suppression dependentupon spatial segregation (REF. 105). Of note, interventionsthat restore the tissue microarchitecture can have impor-tant immune restorative functions104,106. Interestingly,recent studies suggest another instance for which the

Figure 5 |Lipid microdomains during Leishmaniainfection of macrophages.

During the initial encounter, lipid microdomains on the plasma membrane of the

macrophage have a role in directing parasite uptake and the entry of specific

virulence factors such as major surface protein (MSP, also known as GP63). Virulence

factors can also be transferred to the macrophage by parasite-produced exosomes.

Once the promastigote has entered the phagosome, lipophosphoglycan (LPG) inserts

into lipid rafts and inhibits phagosomelysosome fusion. Additional metacyclic pro-

mastigote-derived virulence factors may cross the phagosome membrane using lipid

microdomains or by exosomes, and thereby reach their cytosolic targets. Similar rolesfor lipid microdomains could be postulated for phagosomes containing amastigotes,

although little data exist to directly support this. Altered lipid rafts may also be

responsible for defective antigen presentation and CD40 signalling. MHC class II,

major histocompatibility complex class II.

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DC

DC

Monocyte

DC

CD4+ CD8+

CD8+

Macrophage

Central memoryEffector cells

IL-10

Antigen presentation

IFN

TReg

B cell

TH1

NK cell

Macrophage

Concomitant immunity

A situation in which

immunological resistance to

reinfection co-exists at the

same time as persistence of

the original infection.

mechanism behind an ineffective immune responseand, hence, enhanced parasite persistence could onlyhave been uncovered by in situ approaches. The interac-tions between CD4+T cells and L. major-infected hostcells have been visualized using two-photon imaging inestablished dermal lesions. Although many of the specificT cells were found to colocalize with parasite-infectedcells, there were areas of tissue where accumulations ofparasites were ignored by T cells107. Why this is the caseremains to be determined. However, avoidance of detec-tion at this level does not seem to occur in the case ofhepatic L. donovaniinfections, in which parasites arefound in defined granulomas and infiltrating effectorCD8+T cells appear to be readily able to seek out theirtargets8.

As mentioned earlier, there have been many mecha-nisms described by which parasites attempt to modulate

the ability of their host cells to respond to the signalsthat lead to induction of leishmanicidal activity. Fromthese observations one might conclude that completelyeliminating parasites may not be possible regardless ofthe magnitude of the immune response. However, earlierstudies in which IL-10 was eliminated have suggested thatsterile cure could be achieved in certain situations92. Morerecently, in mice lackingBCL-2-interacting mediator ofcell death (BIM), a pro-apoptotic BCL-2 family member,not only did L. majorinfections resolve, but also this reso-lution was associated with the clearance of all detectableparasites108. Hence, given a sufficient immune response,sterile cure can be achieved in leishmaniasis.

Memory and vaccination.As parasites generally per-sist, even after apparent clinical cure, the type of immu-nity that is induced by Leishmaniainfections is akin toconcomitant immunity. The T cell subsets that contributeto such immunity have been characterized over the pastseveral years, and include CD4+T cells with a phenotypeof central memory T cells109, effector T

H1 cells and resting

effector TH1 cells110. In addition, CD8+T cells can have a

critical role in resistance to reinfection85. The question ofhow to generate these cells by vaccination remains a chal-lenge, as does the question of which T cells may be mostlikely to survive and provide the best protection. Recentstudies suggest that the most protective CD4+T cells arethose that are multifunctional, capable of producing notonly IFN, but also IL-2 and TNF111. Consistent with thefindings that elimination of IL-10 can promote resistance,IL-10 appears to limit the generation of these protective

T cells during vaccination112. Unfortunately, althoughseveral approaches have been taken to develop a vaccinefor leishmaniasis, to date none have been successful inhumans (reviewed in REFS 1,113).

Concluding remarks

Challenges remain in understanding leishmanial biol-ogy, the host responses to the parasites and how to usesuch knowledge to develop new ways of combating theinfection. However, advances over the past several yearsprovide a roadmap for future discovery. The sequenc-ing of several leishmanial genomes provided a wealthof information that will be used to define new drug

Figure 6 |Cellular components of the anti-leishmanial immune response. Monocytes infiltrate the site of

infection and differentiate into dendritic cells (DCs). DCs become infected but fail to become activated, whereas

local uninfected DCs upregulate major histocompatibility complex class II (MHC class II). Macrophages are also

infected by the parasites. Uninfected DCs may pick up dead parasites or leishmanial antigen and become the critical

antigen-presenting cells (APCs). CD4+T cells are then activated and differentiate into T helper (TH1) cells, which

produce interferon-(IFN), and this promotes parasite killing by infected cells and also further promotes thedevelopment of T

H1 cells. Some CD4+T cells fail to become T

H1 cells, and adopt a central memory T cell phenotype.

CD8+T cells recognizing leishmanial antigens are also activated and also produce IFN. Control of the response islargely mediated by the production of interleukin-10 (IL-10), which can come from several different cell types,

including regulatory T (TReg

) cells, TH1 cells, CD8+cells, natural killer (NK) cells, B cells, macrophages and DCs.

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targets, mechanisms responsible for drug resistanceand virulence factors. Species diversity within the genusLeishmaniahas a major role in the manifestations of thedisease. Thus, the application of genomic approaches,such as the rapid sequencing of parasite genomes as wellas omics analyses of the parasite and the host cell duringinfection, will certainly lead to a better understanding ofthe pathogenesis of all forms of leishmaniasis.

A fresh look at the monocyte, macrophage andDC subsets that have central roles in the aetiologyof Leishmania infections, is likely to yield importantadvances that will lead to new ideas for treatment. Thequest for a prophylactic vaccine continues, although

whether it is possible to develop a vaccine that pro-vides long-term protection without constant boostingremains unknown. Nevertheless, harnessing the immuneresponse to control infection will require a better under-standing of the various types of T cells that promoteprotection as well as pathology and that regulateimmunity to the parasite. Finally, Leishmania spp. willcontinue to provide powerful tools to interrogate theimmune system, in exploring the function of myeloid-lineage cells, understanding the role of innate immuneresponses in both protection and pathology and defin-ing the development and interactions between effector,regulatory and memory T cells.

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AcknowledgmentsWork in the P.K. laboratory is funded by grants from the UK

Medical Research Council and the Wellcome Trust. Work in the

P.S. laboratory is funded by grants from the National Institute

of Allergy and Infectious Diseases, US National Institutes of

Health.

Competing interests statementThe authors declare no competing financial interests.

FURTHER INFORMATIONPaul Kayes homepage:

http://www.york.ac.uk/cii/staff/academic/kaye/

Philip Scotts homepage: http://www.med.upenn.edu/apps/

faculty/index.php/g20001882/p4382139

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