Evasion of innate immunity by parasitic protozoa

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www.nature.com/natureimmunology • november 2002 • volume 3 no 11 • nature immunology 1041

Parasitic protozoa are a major cause of globalinfectious disease. These eukaryotic pathogenshave evolved with the vertebrate immune systemand typically produce long-lasting chronic infec-tions. A critical step in their host interaction isthe evasion of innate immune defenses.The abili-ty to avoid attack by humoral effector mecha-nisms, such as complement lysis, is of particularimportance to extracellular parasites, whereasintracellular protozoa must resist killing by lyso-somal enzymes and toxic metabolites.They do soby remodeling the phagosomal compartments inwhich they reside and by interfering with signal-ing pathways that lead to cellular activation. Inaddition, there is growing evidence that proto-zoan pathogens modify the antigen-presentingand immunoregulatory functions of dendriticcells, a process that facilitates their evasion ofboth innate and adaptive immunity.

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.Correspondence should be addressed to D. S. ([email protected]).

Evasion of innate immunity by parasitic protozoa

David Sacks and Alan Sher

Parasitic protozoa are unicellular eukaryotic pathogens that dwellinside host cells and/or in extracellular fluids. These organisms typical-ly produce long-lasting chronic infections in order to maximize theiropportunities for successful transmission by contact with their interme-diate hosts or release into the environment. Their success as parasitesdepends on a series of intricate and highly evolved host adaptations thatenable them to evade destruction by the immune system.

Protozoan infections are a major global health problem, affectingover half a billion people world wide. Several of the diseases theyinduce (such as malaria, African trypanosomiasis and visceral leishma-niasis) represent major causes of mortality and morbidity in tropicalcountries and, as such, are important impediments to economic devel-opment. No standardized vaccines exist for preventing any of the pro-tozoan infections of humans, a situation attributed both to the com-plexity of the pathogens involved and their sophisticated strategies forevading host immune responses1.

Protozoan parasites are highly adept at escaping the effects of adap-tive humoral and cellular immunity. Perhaps the simplest solution forevading antibody responses is the adoption of an intracellular life style,as is done by Leishmania, Trypanosoma cruzi, Toxoplasma gondii andthe liver and blood stages of malaria. Another major strategy—anti-genic variation—protects extracellular protozoa such as African try-

panosomes2 and Giardia3 and even malaria parasites4, which expresstheir antigens on the surface of infected erythrocytes, from immunerecognition. In addition, there is abundant evidence that protozoaninfections actively regulate adaptive T cell responses, resulting in sup-pressed effector functions1. A striking example of this phenomenon isthe recent demonstration that Leishmania major actively induces inter-leukin 10 (IL-10)–producing CD25+ T regulatory cells to prevent com-plete clearance of the parasite5.

Although parasitic protozoa have provided some of the best stud-ied paradigms of evasion of antibody- and T cell–mediated immunityby pathogens, a series of equally important adaptations occur duringthe initial establishment of infection, when parasitic invaders confrontthe innate immune system. The innate host defenses that the majorblood and tissue protozoan pathogens of humans must penetrate,modify or avoid vary according to their mode of entry and the prima-ry host tissue encountered (Table 1). These innate defenses includethe epithelial barrier of the skin, the alternative complement cascadeand other lytic serum components, lysosomal hydrolases and toxicoxygen metabolites of mononuclear phagocytes and the antigen-pre-sentation and immunoregulatory functions of dendritic cells (DCs),which provide a crucial link with the adaptive immune response6.That protozoa have evolved specific mechanisms to evade thesedefenses is dramatically shown by the rapid destruction of preinfec-tive vector-derived life cycle stages when artificially inoculated intomammalian hosts. These developmental adaptations, which enableblood and tissue protozoa to “run the gauntlet” of the vertebrateinnate response, are the focus of our review.

Evasion of humoral innate defensesInnate resistance to protozoa is mediated, in part, by pre-existing sol-uble factors that can potentially recognize and destroy invading para-sites or target them for killing by effector cells. The alternative path-way of complement activation provides a first line of defense againstextracellular parasites that must be subverted for infection to proceed.For example, whereas the epimastigote stage of T. cruzi found ininsect vectors is susceptible to the alternative complement pathway,infective metacyclic and blood-stream trypomastigotes are resistant7.In this case, evasion of complement appears to be due to trypo-mastigote expression of a 160-kD glycoprotein (gp160), which is ahomolog of the host complement-regulatory protein decay-accelerat-ing factor (DAF)8. Like DAF, gp160 can bind to C3b and C4b andinhibit the uptake of subsequent members of the complement cascade,thus preventing convertase formation and lysis of the parasite.Importantly, whereas complement-sensitive epimastigotes fail toexpress gp160, epimastigotes transfected with gp160 are resistant tocomplement-mediated lysis9.

Another interesting strategy is deployed by Leishmania species, forwhich the infective insect stage is transiently exposed to potentially

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nature immunology • volume 3 no 11 • november 2002 • www.nature.com/natureimmunology

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lethal serum components after inoculation by pool-feeding phle-botomine vectors. Leishmania evade complement-mediated lysiswhile using complement activation as a mechanism for targeting hostcells. When insect-stage procyclic promastigotes develop into infec-tive metacyclic forms, their membrane is altered to prevent insertionof the lytic C5b-C9 membrane attack complex (MAC)10. This corre-lates with their expression of a modified surface lipophosphoglycan(LPG) that is approximately twice as long as the form on procyclicpromastigotes, and which may act as a barrier for insertion of theMAC into the parasite surface membrane11. A further developmentalchange that occurs during generation of metacyclics is enhanced syn-thesis of the surface proteinase gp63, which can cleave C3b to theinactive iC3b form, thus preventing deposition of the lytic C5b-C9complex12. However, iC3b also opsonizes the parasites for phagocyto-sis through the complement receptors CR3 and CR1, thereby targetingthe parasite to the macrophage, its host cell of choice13. Conclusiveevidence that LPG and gp63 are virulence factors has emerged fromstudies with L. major null-mutants that do not express these mole-cules; in each case these mutants were highly sensitive to complement-mediated lysis and showed reduced virulence in BALB/c mice14,15.

In addition to complement, protozoa must evade other solublemediators of innate immunity in order to establish a foothold in thehost. Perhaps the best studied are the primate-specific trypanosomelysis factors (TLFs)16, which contribute to the resistance of humans toinfection with Trypanosoma brucei, an important pathogen of live-stock. Biochemical analysis of the activity present in human serumrevealed that high-density lipoproteins are part of the substance thatmediates cytolysis of the parasite, and initial studies demonstratedthat this complex, TLF1, is composed of several common apolipopro-teins as well as a haptoglobin-related protein (Hpr)17,18. A secondcytolytic complex, TLF2, has also been identified that shares many ofthe components of TLF1 but contains a distinct immunoglobulin Mcomponent and a lower lipid content19. Although the final lytic eventhas not been delineated, TLF must undergo receptor-mediated uptakeand enter an intracellular acidic compartment for cytotoxicity tooccur18. Whereas T. brucei is sensitive to TLF-mediated killing, thespecies that infect humans—T. brucei gambiense and T. brucei rhode-siense—are both refractory. This resistance is associated with a blockin TLF endocytosis and retention of the complex in a structure knownas the flagellar pocket16. The refractoriness of certain T. brucei rho-densiese strains to lysis also correlates with the expression of a serumresistance–associated (SRA) gene that is equivalent to truncated vari-ant surface glycoprotein20. Importantly, transfection of SRA from T.brucei rhodesiense into T. brucei confers resistance to lysis by humanserum; this argues that its expression may have been a critical step inthe adaptation of an ancestral parasite for infection of primates21.Interestingly, antibodies raised against a homolog of SRA localize tothe flagellar pocket, suggesting a possible role for the protein in TLFretention22.

Remodeling host-cell compartmentsParasitic protozoa that are adapted to an intracellular lifestyle mustresist the antimicrobial mechanisms that can be induced in phagocyticand even nonphagoctyic host cells. Insofar as the acidified hydrolyticenvironment of host cell lysosomes represents the heart of the defensivemachinery of many nucleated cells, the ability of acid-labile protease-sensitive parasites to avoid or modify this compartment is likely to beone key to their survival (Fig. 1). T. gondii resides in a phagosome thatrestricts its fusion with host endosomes and lysosomes. Toxoplasmaactively penetrates both phagocytic and nonphagocytic cells, propelledby an actin-myosin–dependent gliding motility23. In the process, itestablishes a nonfusigenic compartment, called the parasitophorousvacuole (PV), that lacks integral membrane proteins of host cell origin,but is extensively modified by secreted parasite proteins24,25. Thisremodeling seems to be crucial to the inhibition of PV acidification andlysosome fusion, as these events proceed normally after uptake of deador opsonized parasites, which are internalized via classical receptor-mediated phagocytosis.

T. cruzi trypomastigotes also actively invade mammalian cells but,unlike T. gondii, their cell entry is dependent not on the propulsiveproperties of the parasite, but on their ability to subvert a Ca2+-regulat-ed lysosomal exocytic pathway23. The membrane of the para-sitophorous vacuole is, therefore, derived from the membrane of lyso-somes, and the vacuole itself remains acidic and potentially fusigenicwith other lysosomes. T. cruzi growth and development cannot be sus-tained within this compartment; it depends instead on the ability of theparasite to escape rapidly into the cytosol. Exit from the vacuole ismediated by a parasite-secreted molecule, Tc-TOX, which has mem-brane pore-forming activity at acidic pH and is facilitated by a trans-sialidase present on the trypomastigote surface26,27. Interestingly, T. cruzi is unable to invade cells lacking transforming growth factor-β(TGF-β) receptors I or II28; this suggests that triggering this signalingpathway, perhaps by some parasite TNF-β homolog, might be neededto deactivate host cells during the early stages of infection.

Leishmania parasites lack the machinery necessary for active inva-sion and are confined instead to professional phagocytes, mainlymacrophages, with some exceptions (for example, fibroblasts, DCs andneutrophils)29. The uptake of Leishmania by macrophages proceeds viaconventional receptor-mediated phagocytosis that involves a diversityof opsonic or pattern-recognition receptors (for example, CR3, CR1and mannose fucose receptors)30 that are used depending on the speciesand stage of parasite and the presence or absence of fresh serum.Leishmania metacyclic promastigotes and amastigotes do not seem toremodel the phagosome in a major way because it rapidly fuses (with-in 30 min) with late endosomes or lysosomes31, generating a para-sitophorous vacuole that maintains an acidic pH and hydrolytic activi-ty. Using macrophage cell lines and unselected promastigotes,researchers have shown that LPG-repeating units can transiently inhib-it phagosome maturation and that this delay may be necessary to allow

Table 1.The major protozoan pathogens of humans

Parasite Vertebrate host habitat Mode of transmission Primary site of host invasion

Malaria Intracellular (erythrocytes + hepatocytes) Vector-born (mosquitoes) Skin, blood (sporozoites)Leishmania Intracellular (macrophages) Vector-born (sand flies) SkinToxoplasma Intracellular (macrophages, other Ingestion of parasite cysts Intestinal epithelial cells

nucleated cells)T. cruzi Intracellular (macrophages, muscle cells, Vector-born (reduviid bugs) Skin, conjunctival mucosa

other nucleated cells) and extracellularAfrican trypanosomes Extracellular Vector-born (tse-tse flies) Blood

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sufficient time for promastigotesto differentiate into more hydro-lase-resistant amastigotes32,33.These findings are consistent withthe attenuation of L. major pro-mastigote survival within macro-phages after infection with target-ed null mutants lacking LPG14.The general significance of thetransient inhibition of phago-some-endosome fusion has beenquestioned, however, for two rea-sons. First, Leishmania mexicanapromastigote mutants deficient inLPG or other phosphoglycan-con-taining molecules survive equallywell as wild-type organisms with-in macrophages34, and second,there is an absence of direct datato indicate that mature promastig-otes are any less hydrolase-resis-tant than amastigotes. Thatamastigotes are remarkably robustis clear: the parasite does notescape from the vacuole, but man-ages to withstand the low pH andonslaught of acid hydrolases, pre-sumably by producing an abun-dance of cell surface and secretedglyconjugates that protect the cellfrom proteolytic damage. As theL. mexicana phosphoglycan–deficient mutants were able to survivenormally, it is likely that this species displays different or redundantvirulence determinants. It is possible, for example, that the huge para-sitophorous vacuole that is formed after infection with parasites of theL. mexicana complex might effectively dilute the hydrolases to whichthe parasite is exposed within the vacuole and obviate the need for cer-tain surface or secreted glycans.

Inhibition of host cell signaling pathwaysMacrophages possess primary defense mechanisms—including activa-tion of macrophage oxidative metabolism and synthesis and release ofarachidonic acid metabolites—that are induced by the attachment andengulfment of microbial agents. The major source of reactive oxygenintermediates (ROIs) in macrophages is the NADPH oxidase, a multi-component enzyme that catalyzes the transfer of electrons fromNADPH to molecular oxygen, resulting in the production of superox-ide and hydrogen peroxide35. It is generally believed that activation ofprotein kinase C (PKC) and protein tyrosine kinases (PTKs) are the twocritical events involved in regulating phagocyte functions in response toa variety of extracellular stimuli. In vitro studies involving human cellshave shown that macrophage functions, including phagocytosis andROI generation, are severely impaired after uptake of an insolubledegraded host hemoglobin, called hemozoin, generated during blood-stage malarial infection36 (Fig. 2). These dysfunctions were attributedto inhibition of PKC translocation and activation in the hemozoin-loaded cells37. Early studies suggested that Leishmania parasites alsoavoid triggering the oxidative burst by actively inhibiting PKC activa-tion in macrophages38,39. Inhibition of PKC-mediated protein phospho-rylation was observed with purified LPG40, which might act either as

competitive inhibitor of the PKCactivator DAG and/or by alteringthe physical properties of thebilayer to inhibit PKC membranetranslocation. These findings areagain consistent with impairedintracellular survival of the L.major LPG–deficient mutantcompared to the mutant in whichLPG expression was restored,although effects on specific sig-naling events were not directlycompared14.

In addition to their innatemicrobicidal responses, macro-phages can initiate the host acti-vation cascade by presenting anti-gens and costimulatory moleculesand by providing regulatorycytokines to T cells. A number ofstudies suggest mechanisms bywhich intracellular protozoa caninterfere with the immune-initia-tion functions of macrophages.One of the more consistent andstriking dysfunctions observed inmacrophages infected with proto-zoan parasites is their inability toproduce IL-12, which—as themain physiological inducer ofinterferon-γ (IFN-γ) and T helper

type 1 (TH1) cell differentiation—is an essential cytokine for the devel-opment of acquired resistance to most intracellular pathogens. In addi-tion, excess IL-12 production can lead to severe tissue damage and evenmortality, a consequence that may also be detrimental to the parasite inblocking its transmission cycle. It is no wonder then that protozoa haveevolved a series of intricate and often redundant mechanisms for regu-lating IL-12 production by antigen-presenting cells, especiallymacrophages, which are the major potential source of the cytokinestimulated during early infection.

Infectious stages of Leishmania do not merely avoid IL-12 induc-tion, they actively and selectively inhibit it, leaving other pro-inflam-matory cytokine or chemokine response pathways relatively intact41.Whereas the glycophosphatidylinositol (GPI) anchors of many para-sites are potent macrophage activators, Leishmania LPG and gly-coinositol phospholipid (GIPL) inhibit IL-12p40 transcription whilefailing to suppress TNF gene expression42. Although the receptors andsignaling pathways involved in this selective inhibition of IL-12 syn-thesis have yet to be identified, suppression of NF-κB activity does notappear to be involved. Because many IL-12 agonists that are inhibitedin Leishmania-infected macrophages—including lipopolysaccharide(LPS), CD40 ligand (CD40L) and especially IFN-γ—signal primarilythrough protein-tyrosine kinases, the observation that Janus kinase–sig-nal transducers and activators of transcription (Jak-STAT) signalingpathways are also inhibited in Leishmania-infected cells seems espe-cially relevant to the defective IL-12 response43. Defective phosphory-lation of Jak2 is attributed to rapid activation of a cytoplasmic proteintyrosine phosphatase (PTP), SHP-144 (Fig. 2). SHP-1 is necessary for L. major survival insofar as SHP-1–deficient motheaten mice do notproduce lesions and their macrophages fail to support infection in

Figure 1. Remodeling of macrophage intracellular compartments byparasitic protozoa. T. cruzi trypomastigotes enter the macrophage by induc-ing the recruitment of lysosomes to the plasma membrane; they only transient-ly reside in the parasitophorous vacuole before escape into the cytoplasm viasecretion of a pore-forming molecule, termed Tc-TOX (yellow). T. gondii tachy-zoites actively invade the cell and remodel a parasitophorous vacuole membrane(blue) that contains secreted parasite proteins but excludes host proteins thatwould normally promote phagosome maturation, thereby preventing lysosomefusion. Leishmania metacyclic promastigotes are taken up by receptor-mediatedphagocytosis; phagosome maturation may be transiently inhibited by LPG(green), which becomes incorporated into the phagosome membrane.The repli-cating amastigote stage ultimately resides within a phagolysosome where theysurvive via production of cell-surface and secreted glycoconjugates, includingGIPLS and proteophosphoglycan (PPG) (green).

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vitro45. Because specific ligation of the macrophage receptors CR1 andCR3 also leads to selective inhibition of IL-12 production—which, atleast in the case of CR3, is associated with impaired tyrosine phospho-rylation of STAT146—it is likely that host cell PTPs are rapidly inducedby CR3 ligation during attachment and uptake of serum-exposed C3-opsonized parasites.

Given that Jak-STAT signaling pathways are involved in a broadrange of macrophage responses, including induction of most pro-inflammatory cytokines, it is difficult to understand how Leishmaniainfection maintains such a selective effect on IL-12. One possibleexplanation lies in two observations: first, STAT1 regulates IFN con-sensus-binding protein (ICSBP) induction and—of the cytokinesassayed—only activation of the IL-12p40 promoter appears to requireICSBP, and second, ICSBP knock-out mice (on resistant background)are susceptible to L. major infection47. The involvement of redundantSTAT1-independent induction pathways for tumor necrosis factor-α(TNF-α), IL-1β and inducible nitric oxide synthase (iNOS) has beendescribed (see below) and suggests a basis for the maintenance of theseresponses in Leishmania-infected cells. A similar mechanism mayaccount for the down-regulation of IL-12p40 gene expression in mousemacrophages after uptake of Plasmodium berghei–infected erythro-cytes48, which appear to be selective for IL-12 also and do not appear tobe due to impaired NF-κB activation.

The NF-κB family of transcription factors is an evolutionarily con-served group of proteins that are important in the regulation of numerousgenes involved in innate and adaptive immunity—including those encod-ing IL-12, IFN-γ, TNF-α, iNOS and adhesion molecules—as well asthose involved in cell proliferation and survival. Studies in T. gondii49,50

have demonstrated the ability of intracellular protozoa to actively interferewith the NF-κB–activation pathway in macrophages. In T. gondii–infect-ed cells, despite rapid IκB phosphorylation and degradation, NF-κB failsto translocate to the nucleus. In contrast to Leishmania, T. gondii–inducedresponse defects are more generalized and include both IL-12 and TNF-α. With the use of virulent strains of T. gondii, the inhibition of NF-κBtranslocation in macrophages and its effect on iNOS expression wasshown to be dependent on HS70 expression by the parasite that mightimpede nuclear transport by competing for access to nuclear pore com-plexes51. Disrupted nuclear transport of phosphorylated STATα has alsobeen reported for T. gondii–infected macrophages and is thought to inhib-it Jak-STAT–dependent MHC class II expression51 (Fig. 2).

In addition to being directly suppressed by the parasite within infect-ed cells, macrophage IL-12 production and effector functions can alsobe held in check by the down-modulatory cytokines IL-10 and TGF-β,which can themselves be up-regulated by the parasites or their prod-ucts. Striking evidence in support of the importance of IL-10–depen-dent down-regulation of IL-12 production for both host and parasitecomes from studies in IL-10–deficient mice infected with either T.gondii or T. cruzi. These animals display dysregulated IL-12 productionand die of cytokine-associated tissue damage while showing decreasedparasite burdens52,53. Similarly, low amounts of active TGF-β produc-tion by splenic mononuclear cells are associated with a lethal outcomein rodent malaria, and anti–TGF-β treatment transformed a normallyresolving infection into a lethal one due to overproduction of patho-genic pro-inflammatory cytokines54. With respect to Leishmania, thereis as yet no evidence that deficient production of these anti-inflamma-tory mediators is responsible for severe clinical outcomes. In contrast,there is ample evidence to suggest that overproduction of thesecytokines contribute to uncontrolled parasite growth and nonhealinginfections. Both IL-10 and TGF-β are produced by murinemacrophages after Leishmania infection in vitro, and both promoteintramacrophage replication and are important factors for determiningin vivo susceptibility55,56.

Finally, parasitic protozoa have evolved strategies to down-modulatesignaling pathways leading to host cell apoptosis, thereby prolongingthe life of the host cell and their own intracellular survival57. Inducedapoptotic pathways in macrophages infected with either Leishmaniadonovani promastigotes58 or T. gondii tachyzoites59 were strongly inhib-ited, perhaps by parasite induced up-regulation of Bcl-2 homologs.Interestingly, the pro-apoptotic effects that T. cruzi infection has onboth CD4+ and CD8+ T cells indirectly enhanced parasite growth, as thebinding of apoptotic lymphocytes to vitronectin receptors onmacrophages triggered TGF-β production and a burst of parasite repli-cation in infected cells60.

Manipulation of DC functionTo the extent that pathogenic protozoa have learned to condition theirinitial encounter with the innate immune system as a means of manip-ulating the subsequent adaptive immune response, it has been impor-tant to extend these observations to effects on “professional” antigen-presenting cells. In contrast to primary targets of infection, such as

Figure 2. Inhibition of macrophage signalingpathways. Inhibition of pathways by malaria-gener-ated hemozoin or infection with Leishmania or T.gondii. Hemozoin and Leishmania impair the oxidativeburst associated with phagocytosis by inhibiting thePKC activation required for assembly of the NADPHoxidase complex in its active form. Leishmania para-sites also inhibit IFN-γ– or CD40L-induced PTK-dependent signaling involved in IL-12 production byactivation of the cellular phosphatase SHP-1 thatinhibits Jak2 and STAT1 phosphorylation (P).Toxoplasma inhibits LPS-induced cytokine responsesby inhibiting nuclear translocation of NF-κB and pos-sibly phosphorylated STAT1.

RhoGDI

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macrophages, DCs may be temporally removed from encounter withparasites or their products and may respond distinctively. For example,whereas IL-12 production is actively suppressed in macrophagesinfected with L. major or T. gondii, DCs actively produce IL-12p40 inresponse to the same parasites, both in vitro and in vivo61–65. It should benoted that in each case the parasite infection alone was not sufficient forhigh IL-12p70 production in vitro and that endogenous agonists, suchas CD40L or IFN-γ, were necessary as costimuli. The different out-comes of protozoan encounter on IL-12 production by macrophagesversus DCs is consistent with the delayed, albeit effective, immuneresponse to these infections that is ultimately achieved. Apart from theobvious parasite advantage that delayed onset of immunity brings tobear, suppression of IL-12 production by infected macrophages mayagain represent a coadaptation that dampens their local activation whileprotecting the host from cytokine shock.

For those parasitic infections associated with T cell control mecha-nisms that are not simply delayed, but are persistently compromised,impaired maturation and function of appropriate DC subsets might bemore expected (Table 2). A particularly interesting example is the inhi-bition of DC maturation by the malaria parasite Plasmodium falci-parum66. Malaria-infected erythrocytes bind to the surface of myeloidDCs in vitro and markedly suppress the normal up-regulation of majorhistocompatibility complex (MHC) class II molecules, adhesion mole-cules (for example, ICAM-1) and costimulatory molecules (CD83 andCD86) on the cells after stimulation with LPS. The resulting DCs wereseverely impaired in their capacity to induce allogenic as well as anti-gen-specific primary and secondary T cell responses67. The receptors onthe surface of DCs mediating this inhibitory effect are CD36 and CD51,and their artificial ligation mimicked the suppression induced by infect-ed erythrocytes68. Interestingly, the same receptors are also involved inthe recognition of apoptotic cells and, when incubated with DCs, suchcells trigger reduced secretion of IL-12 and increased production of IL-10 by these antigen-presenting cells. The major parasite ligand forCD36 binding in infected erythrocytes is thought to be a conserveddomain of P. falciparum erythrocyte membrane protein 1 (PfEMP1), amolecule that undergoes antigenic variation. Thus, a nonadherent para-site line, which does not express PfEMP1 antigens on the red cell sur-face, failed to inhibit DC maturation.

The relevance of these observations to acute disease is indicated bythe finding that the percentage of HLA-DR+ DCs was significantlylower in children with severe or mild malaria compared to healthy con-trols68. At present there is no direct evidence that impaired DC matura-tion in children with acute malaria is CD36-mediated or that this eventis at all necessary for successful malaria infection. Nevertheless, it isintriguing that infected erythrocytes with high CD36 binding affinityare more frequently observed in isolates from patients with mild asopposed to severe disease69,70. It suggests that adhesion of infected ery-throcytes to CD36 may suppress the pro-inflammatory response to theparasite, an outcome that would favor both host and parasite.

Infection of monocyte-derived human DCs with T.cruzi trypomastigotes alsoinhibits LPS-induced secre-tion of pro-inflammatorycytokines—including IL-12and TNF-α—as well as HLA-DR and CD40 up-regulation71.Similar effects were observedwhen human DCs wereexposed to T. cruzi–derived

GIPL or to only the ceramide portion of the compound72, althoughnonphysiological high concentrations of both molecules were used inthis in vitro study.

With respect to Leishmania, the spontaneous migration of murinesplenic DCs or Langerhans cells (LCs) was inhibited by L. major pro-mastigote–conditioned media or L. major LPGs, respectively; this sug-gested that their ability to transport antigen to or within lymphoid tis-sue might be impaired during infection73,74. These effects need to beinterpreted, however, in the context of other studies (referred to above).These studies involved infection of murine DCs with viable L. majorpromastigotes or amastigotes; the infected cells up-regulated MHCclass II and costimulatory molecules, produced IL-12p40 and downregulated E-cadherin, which indicated that they were competent forantigen transport and presentation61,75. Uptake of Leishmania amazo-nensis amastigotes or metacyclic promastigotes by mouse bone mar-row–derived DCs (BMDCs) also induced up-regulated expression ofMHC class II, CD40, CD80 and CD86 but, in contrast to L. major, theinfected DCs produced IL-4 and no IL-1276. This suggested that theDCs were conditioned by the parasite to prime the pathogenic TH2 cellsthat dominate the response in vivo.

Studies involving yet other Leishmania species suggest a more pas-sive strategy for immune evasion, in which their interactions with anti-gen-presenting cells appear to proceed in a relatively silent fashion.Uptake of L. mexicana by mouse BMDCs was not sufficient to activatethem in vitro, although their maturation response to other stimuli wasnot impaired77. A similar species restriction in Leishmania-induced DCactivation was observed after uptake of metacyclic promastigotes byhuman myeloid DCs, which were efficiently primed for CD40L-induced ILp70 secretion by L. major, but not by L. tropica or L. dono-vani78. These observations may be significant because whereas L. majorproduces self-limiting infections that are controlled in the skin, theother species mentioned are capable of disseminating to the viscera orto other cutaneous sites. Given the well described polymorphisms in thesurface and secreted glycan structures that are expressed by differentLeishmania species79, it will be interesting to determine whether theyaccount—at least in part—for the differences in innate response pat-terns and the spectrum of clinical disease.

From the above discussion it would appear that parasites carefullyregulate the induction of IL-12 from DCs during early infection as ameans of determining the character of the adaptive immune responsethat eventually decides their fate in the host. In addition, there is emerg-ing evidence for the existence of distinct parasite-triggered pathwaysfor regulating DC IL-12 production once it has been initiated. One suchmechanism has been identified by studies of IL-12 synthesis by murinesplenic DCs after injection of a soluble extract of T. gondii tachyzoites(STAg). The response was short-lived and could not be recalled by asecond injection of STAg for a period of 1 week after initial priming80.This “paralysis” of the DC IL-12 response induced by STAg does notrequire IL-10 but instead appears to depend on the induction of lipoxin

Table 2. Impairment of DC function by parasitic protozoa

Parasite Inhibitory signal Immunologic effects

Leishmania Live infection (L. amazonensis) Inhibition of DC IL-12 production with turn on of IL-476

Parasite culture media or LPG Inhibition of DC or LC migration73,74

T. cruzi Live infection GIPL (or ceramide portion) Suppression of LPS-induced IL-12,TNF-α , HLA-DR and CD40 expression in human DCs71,72

T. gondii Parasite induction of host LXA4 in vivo Inhibition of CCR5-dependent IL-12 production by DCs80,81

P. falciparum Ligation of CD36 (or CD51) by parasitized Suppression of DC maturation and function67,68

erythrocytes

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A4 (LXA4), a product of arachadonic metabolism81. This eicosonoidappears to function by suppressing CCR5 expression, a chemokinereceptor involved in IL-12 stimulation by STAg. The same mechanismof IL-12 suppression is likely to operate during natural infectionbecause T. gondii–infected mice with a defect in an enzyme (5-LO)required for LXA4 show excess IL-12 production and, in common withinfected IL-10–deficient mice53, succumb to cytokine shock and showdecreased parasite burdens (J. Aliberti et al., unpublished data). Takentogether, these findings suggest that once IL-12 production by DCs hasbeen induced and TH1 responses initiated, synthesis of the cytokine isactively down-regulated, probably to the joint benefit of both host andparasite. Whether analogous mechanisms suppress the IL-12 responsesof DCs to other protozoa remains to be determined.

Concluding commentsIn learning to evade innate host defenses, protozoan parasites appear tohave mastered the intricacies of both cell biology and cellularimmunology. As such, they can teach us important lessons about bothfields of biology as well as their points of intersection. In particular, thestrategies used by these pathogens for remodeling cellular compart-ments offer insights into the dynamics of host membranes and intracel-lular trafficking. At the same time there is much to be learned abouthost-signaling pathways and immune-response initiation and polariza-tion by studying the mechanisms used by protozoa to suppress or divertimmune responses. Because innate defenses (for example, lysis bycomplement or TLF) have the potential to block infection, the mecha-nisms used to evade them are potential “Achilles’ heels”, and offernewly identified and largely unexploited targets for vaccine and/orpharmacologic intervention.

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Evasion of innate immunity by parasitic protozoa