433

Immune responses to protozoans

Peter C. Melby, Gregory M. Anstead 28Protozoal infections are an important cause of morbidity and mortality worldwide ( Table 28.1). Protozoan pathogens exact their major toll in the tropics, but infection with these parasites remains a significant prob-lem in developed countries, owing to travel to and emigration from developing countries, the susceptibility of AIDS patients to opportunis-tic protozoans, and episodic transmission within communities.

Protozoan pathogens make up a group of highly diverse organisms that use a wide array of mechanisms of pathogenesis to evade the host immune response (Table 28.2). There are numerous targets for the intra-cellular protozoan parasites, including erythrocytes (Babesia and Plasmodium), macrophages (Leishmania and Toxoplasma gondii), or mul-tiple cell types (Trypanosoma cruzi). The luminal parasitic protozoan may be extracellular, such as the amebae and the flagellates (Giardia and Trichomonas), or primarily intracellular, such as the coccidian parasite Cryptosporidium.

The innate and acquired immune responses to the protozoan patho-gens are summarized in Table 28.3. Neutrophils and macrophages are the effector cells that mediate the innate response against the extracel-lular protozoan parasites. The natural killer (NK)-cell-activated macro-phage system is central to the innate response to the intracellular para-sites (Fig. 28.1) (Chapters 3 and 18). The innate cytokine response also plays a critical role in the generation of the adaptive immune response. For the intracellular pathogens (e.g., Leishmania spp., T. cruzi, T. gondii), the early production of IL-12 and IFN-γ drives the differentiation of T cells to a protective Th1 phenotype. In most cases CD4 T cells play a primary role in acquired cellular immunity, but CD8 T cells may play a critical role through cytokine production (e.g., Plasmodium spp., T. cruzi, T. gondii) or direct cytotoxic activity (e.g., Cryptosporidium). For the parasites that have an extracellular stage (e.g., Plasmodium spp., the trypanosomes, Giardia and Trichomonas), specific antibodies play a role in the acquired immune response.

Intensive effort has been dedicated to the development of effective vaccines for protozoal diseases, but despite their significance none has so far reached the stage of clinical use. A review of all the potential vaccine candidates is beyond the scope of this chapter and the reader is referred to a number of excellent reviews.1–4 A discussion of the immune responses to some of the individual protozoal pathogens follows.

� PLASMODIUM SPP. �PATHOGENESIS

Soon after sporozoites of the Plasmodium spp. are injected into the bloodstream by the Anopheles mosquito they invade hepatocytes and undergo schizogony (asexual reproduction). A dormant form of P. vivax and P. ovale (hypnozoites) can reside within hepatocytes for months before causing a clinical bloodstream infection. Following schizogony, merozoites are released from ruptured hepatocytes into the bloodstream, where they invade red blood cells to produce ring-stage parasites. These parasites mature into trophozoites, which again undergo schizogony, leading to rupture of the erythrocyte and the release of merozoites. The merozoites then invade fresh red blood cells (RBC), or develop into male or female gametocytes that can then be picked up by another feeding mosquito to continue the transmission cycle.

HOST DEFENSE AGAINST PROTOZOA

>> Interaction of the parasite with host cells induces an array of

cytokines which stimulate the innate and adaptive immune

responses to eliminate the pathogen, and/or cytokines that

inhibit or downregulate the antiparasitic responses to enable

the initiation of tissue parasitism.

>> The outcome of infection is determined by the balance

between the infection-promoting and the host-protective

cytokines and effector cells. Often there is a mixed response,

resulting in a persistent subclinical infection.

>> A persistently infected host may develop clinical disease if

there is a waning of the immune mechanisms (e.g., in AIDS)

that are critical to the control of infection.

KEY CONCEPTS

PL

AS

MO

DIU

M S

PP.

INFECTION AND IMMUNITY

434

28

The clinicopathological features of malaria are caused by the intra erythrocytic stage. Schizogony and rupture of RBCs is associated with fever. Much of the tissue damage is mediated by the adherence of P. falciparum-infected RBCs to endothelial cells through multiple ligand–receptor interactions and plugging of microcapillary beds. Several P. falciparum trophozoite proteins, most notably belonging to the erythrocyte membrane protein-1 (EMP-1) family, interact either directly or indirectly with the RBC membrane, resulting in abnormalities that promote cytoadherence.5, 6 A number of endothelial adhesion molecules, including intercellular adhesion molecule-1, vascular cell adhesion molecule-1, thrombospondin, E-selectin, CD31, CD36, hyaluronic acid, and chondroitin sulphate A, mediate cytoadherence. Sequestration of parasitized RBCs in the capillary beds offers a survival advantage to the parasites by removing them from circulation through the spleen. Along with the sequestered RBCs there is accumulation of intravascular macrophages, neutrophils and platelets.

The induction of a proinflammatory cytokine cascade and counter- regulatory responses plays a central role in the pathogenesis of P. falciparum malaria and its complications. Parasite antigens, particu-larly those having glycophosphatidyl inositol membrane anchors, released during the rupture and reinvasion of RBCs, activate the innate immune response through interaction with receptors on host cells. The production of proinflammatory cytokines (IL-1, TNF-α, IL-12, and IFN-γ), which leads to fever, expression of endothelial adhesion mol-ecules, and cytoadherence, is mediated in part by TLR-2 and is MyD88 dependent.5, 6 The induction of nitric oxide (NO) synthesis by endothelial cells may also contribute to inflammatory lesions in the brain. Cerebral malaria in the mouse model can be prevented by neu-tralization of TNF-α, and the risk of cerebral malaria in children is increased when the child has an allele containing a TNF-α promoter polymorphism that is associated with increased TNF-α transcription.

ParasiteEstimated worldwide cases (annual mortality) Clinical manifestations

Trypanosoma brucei complex 100 000 new cases/year (5000 deaths) Intermittent fever, lymphadenopathy,

meningoencephalitis

Trypanosoma cruzi 24 million (60 000 deaths) Asymptomatic infection; dysrhythmias or

chronic heart failure; hypertrophy and dilation

of the esophagus, colon

Entamoeba histolytica 50 million (100 000 deaths) Asymptomatic infection, diarrhea, dysentery,

or liver abscess

Cryptosporidium parvum Prevalence 3–10% in patients with diarrhea

in developing countries

Self-limited diarrhea in immunocompetent

persons, severe intestinal and biliary disease

in AIDS patients

Cyclospora spp. Prevalence of ~10% in developing countries Relapsing watery diarrhea

Giardia lamblia 200 million (most common in young children

and immunocompromised)

Asymptomatic infection, chronic diarrhea

Isospora belli Incidence unknown, rare in immunocompetent

persons

Self-limited diarrhea in immunocompetent

persons, chronic diarrhea in AIDS patients

Leishmania spp.

10–50 million people infected, 1.2 million new

cases per year

Asymptomatic infection; skin ulcers or

nodules; destructive oropharyngeal lesions;

visceral disease with fever,

hepatosplenomegaly, cachexia,

pancytopenia

Plasmodium spp.

400–490 million (P. falciparum: > 2 million

deaths/year, primarily children)

Fever with potential complications of severe

hemolysis, renal failure, pulmonary edema,

cerebral involvement

Toxoplasma gondii

Several hundred million people infected

worldwide. 5–70% of healthy US adults

are seropositive

Self-limited fever, hepatosplenomegaly;

lymphadenopathy and encephalitis

(reactivation in AIDS patients); congenital

infection, with fetal death, chorioretinitis,

meningoencephalitis

Trichomonas vaginalis 170 million/year Asymptomatic infection, vaginal discharge,

urethritis

Table 28.1 Worldwide significance of the major protozoal infections (Summarized from Markell E, John D, Krotoski W.

Medical parasitology. Philadelphia: WB Saunders, 199944)

PL

AS

MO

DIU

M S

PP.

Immune responses to protozoans 28

435

Mechanism of immune evasion Pathogen

Antigenic variation Plasmodium spp., Trypanosoma brucei, Giardia lamblia,

Trichomonas vaginalis

Entry into red blood cells Plasmodium spp., Babesia microti

Resistance to complement-mediated lysis Leishmania spp., Trypanosoma cruzi, Trypanosoma brucei,

Entamoeba histolytica

Impaired macrophage microbicidal function Leishmania spp., Trypanosoma cruzi, Trypanosoma brucei,

Toxoplasma gondii, Entamoeba histolytica

Impaired antigen presentation to T cells Leishmania spp., Trypanosoma cruzi, Toxoplasma gondii

Synthesis of immunosuppressive mediators

(e.g. IL-10, TGF-β, PGE2)

Leishmania spp., Trypanosoma cruzi, Toxoplasma gondii,

Entamoeba histolytica

Generalized depression of T-cell responses Leishmania spp., Trypanosoma cruzi, Trypanosoma brucei

Expansion of regulatory T-cell population Plasmodium spp., Leishmania spp., Trypanosoma cruzi

Inhibition of phagolysosomal fusion Toxoplasma gondii

Direct cytolysis of host inflammatory cells Entamoeba histolytica

Degradation of host antibodies Entamoeba histolytica, Giardia lamblia, Trichomonas

vaginalis

Table 28.2 Principal mechanisms of immune evasion by protozoal pathogens

PL

AS

MO

DIU

M S

PP.

Table 28.3 Principal mechanisms of innate and acquired immunity to protozoal pathogens

Immune mechanism Pathogen

Innate immune response

Complement-mediated lysis Plasmodium spp., Leishmania spp.

IL-12-dependent NK-cell production of IFN-γ,

leading to macrophage activation

Plasmodium spp. (pre-erythrocytic stage), Leishmania spp.,

Trypanosoma cruzi, Trypanosoma brucei, Toxoplasma gondii

Cross-reactive class II-restricted CD4 T cells Plasmodium spp., Toxoplasma gondii

IFN-γ production by γδ T cells Plasmodium spp., Toxoplasma gondii

Activated polymorphonuclear

leukocytes

Leishmania spp., Entamoeba histolytica, Giardia lamblia,

Trichomnas vaginalis, Cryptosporidium parvum

Interferon-α/β activation of macrophages Leishmania spp.

Antimicrobial peptides (defensins) Giardia lamblia, Cryptosporidium parvum

Toll-like receptor signaling Plasmodium spp., Trypanosoma cruzi, Leishmania spp.

Acquired immune response

Parasite-specific IgG antibody

response

Plasmodium spp., Trypanosoma cruzi, Trypanosoma brucei,

Giardia lamblia, Cryptosporidium parvum

Plasmodium spp., Leishmania spp., Trypanosoma cruzi

Trypanosoma cruzi, Cryptosporidium parvum

Plasmodium spp., Leishmania spp., Trypanosoma cruzi,

Trypanosoma brucei, Toxoplasma gondii, Entamoeba histolytica,

Cryptosporidium parvum

Toxoplasma gondii (cyst stage), Entamoeba histolytica, Giardia

lamblia, Cryptosporidium parvum

Class I-restricted, IFN-γ-producing CD8 T cells,

which activate macrophage microbicidal mechanisms

Class I-restricted cytotoxic CD8 T cells

Class II-restricted, IFN-γ-producing CD4 T cells,

which activate macrophage microbicidal

mechanisms

Mucosal IgA antibody response

INFECTION AND IMMUNITY

436

28

GPI molecules also activate CD1d-restricted natural killer T (NKT) cells, leading to accelerated proinflammatory cytokine synthesis. T cells primed by previous exposure to either malaria or cross-reacting anti-gens also produce IFN-γ, which contributes to the production of a pathologically high level of TNF-α. Downregulation of the Th1 immune response and TNF-α production by IL-10 acts to limit the inflammatory response.

INNATE IMMUNITY

Complement-mediated lysis may occur at the sporozoite and merozoite stages. An IL-12 and IFN-γ-dependent pathway of innate resistance to exoerythrocytic stages of murine malaria has been identified. NK cells are probably the source of the IFN-γ.7 Although high levels of TNF-α are associated with severe malaria, physiological levels are protective through the activation of macrophages that can clear pre-erythrocytic-stage parasites.

The role of T cells in innate immunity to Plasmodium spp. is unclear. Malaria-reactive CD4 T cells, which express the αβ TCR and are MHC class II restricted, have been identified in nonexposed individuals. Additionally, γδ TCR+ T cells that respond to phosphorylated nonpep-tide antigens on live parasites have been demonstrated. Both of these cell populations can secrete IFN-γ, and could therefore lead to phagocyte activation and killing of parasites.

ACQUIRED IMMUNITY

Partial immunity to Plasmodium spp. infection is acquired slowly follow-ing repeated exposure in endemic areas. In areas of intense P. falciparum transmission the density of parasitemia, the morbidity and the incidence of cerebral malaria and malaria-related deaths is highest in the early childhood years, declining thereafter.

Acquired immune mechanisms active at the pre-erythrocytic stage have largely been identified through studies in which mice were vacci-nated with large numbers of irradiated sporozoites and challenged with murine Plasmodium spp. For reasons that are not clear, vaccination with sporozoites in experimental systems induces much stronger immunity than that induced by natural infection. Antibodies directed against a number of antigens expressed on the surface of the circulating sporozoite before it enters the hepatocyte are able to neutralize its infectivity. Anti-sporozoite antibody-mediated immunity requires the presence of high antibody titers to prevent sporozoite invasion of the hepatocyte, which occurs within minutes of inoculation. Acquired immunity to the pre-erythrocytic stage is mediated primarily through class I-restricted para-site-specific CD8 T cells, and to a lesser extent CD4 T cells, via secreted IFN-γ, which induces nitric oxide (NO)-dependent killing of the intra-hepatocyte parasites.8

Both antibody-dependent and cell-mediated (antibody-independent) immune mechanisms are active against the erythrocytic stage of infection.9 Immunity develops following drug-cured blood-stage infection in laboratory animals, and the immunity is stronger after repeated episodes of infection/cure. Adoptive transfer of antibodies from drug-cured mice confers protec-tion to naïve mice. Similarly, adoptive transfer of immune human serum is protective for naïve individuals. Antibodies directed against merozoite sur-face proteins may inhibit invasion. The IgG1 and IgG3 isotypes play a role in naturally acquired immunity by opsonizing schizont- and trophozoite-infected RBCs (recognizing parasite antigens on the surface of the RBC) so that they can be cleared by phagocytic cells, especially in the spleen.

CD4 T cells are also able to confer immunity to blood-stage infection when adoptively transferred from immune mice. The mechanism(s) of CD4 T cell-mediated immunity is unclear, but MHC class II-restricted antigen presentation and T-cell co-stimulation are required, and the gen-eration of cytokines and nitric oxide has been implicated in some studies. Protective cellular immune responses (CD4 and CD8 T-cell proliferation, IFN-γ production and NO synthesis) in the absence of detectable

Macrophageactivated

for intracellularkilling

Macrophageor dendritic cell

IL-10TGF-PGE2

IFN-TNF-

IFN-TNF-

IFN-

IL-12TNF-IL-18IL-1

NK cell(+)

NO(+)

(+)(+)

(-)RNIROI

Pathogen orsoluble products

Fig. 28.1 Macrophage, NK cell and cytokine interactions in the

innate immune response to intracellular protozoa. Exposure of

macrophages or dendritic cells to a pathogen or microbial product

can result in the release of cytokines and inflammatory mediators that

may stimulate (+) or suppress (−) NK cell activation. Activated NK cells

produce cytokines that can then activate macrophages for intracellular

killing. It must be recognized that this diagram is oversimplified and

that these cytokines, most notably IFN-α/β, IL-10, TGF-β, and IL-12,

may be produced by other types of cell, such as epithelial cells or

enterocytes. NO, nitric oxide; RNI, reactive nitrogen intermediates;

ROI, reactive oxygen intermediates.

IMMUNOPATHOGENESIS OF SEVERE

PLASMODIUM FALCIPARUM MALARIA

>> Release of malarial antigens stimulates TNF-α production.

>> TNF-α induces expression of endothelial adhesion molecules

(e.g. intercellular adhesion molecule-1, vascular cell adhesion

molecule-1, thrombospondin, E-selectin, and chondroitin

sulphate A).

>> P. falciparum trophozoite proteins interact with the plasma

membrane of the infected RBC to form knob-like protrusions

and other membrane abnormalities.

>> Infected RBCs with altered membrane surface adhere to

upregulated endothelial adhesion molecules.

>> Trapped infected RBCs are sequestered away from the

splenic defense and cause microcapillary plugging, leading

to inflammatory pathology and possible tissue ischemia.

KEY CONCEPTS

PL

AS

MO

DIU

M S

PP.

Immune responses to protozoans 28

437

antibody responses were identified in naïve volunteers who were repeat-edly exposed to low doses of blood-stage parasites. T cells expressing the γδ receptor have recently been shown to play a role in the control of chronic parasitemia in mice. The T-cell effector mechanisms that are active in acquired immunity to human malaria have not been defined.

EVASION OF HOST IMMUNITY

The malaria parasite uses several different mechanisms to evade the host immune response.5 Sporozoites and merozoites evade circulating antibody by rapidly entering hepatocytes or RBCs, respectively. Mature RBCs do not express MHC molecules on their surface, and so avoid recognition by T cells. The few parasite proteins that are expressed on the erythrocyte surface (e.g., P. falciparum erythrocyte membrane pro-tein-1) exist in multiple allelic forms that have variable B-cell epitopes that induce only variant-specific antibody responses. Many of the immunodominant antigens in Plasmodium spp. are proteins having extensive repeat sequences that vary from strain to strain and tend to function as T-independent antigens that induce short-lived, low-affinity antibodies. Recently, CD4+CD25+ T-regulatory cells were shown to suppress antimalarial T-cell immunity in mice,10 possibly through the production of IL-10 and TGF-β. Both TGF-β, which can be converted from the latent to bioactive form by Plasmodia proteases, and IL-10 may suppress host effector mechanisms. The absence of these counter-regulatory cytokines leads to enhanced inflammatory pathology and mortality in rodent malaria models.

� LEISHMANIA SPP. �PATHOGENESIS

The intracellular Leishmania amastigote replicates within macrophages in the vertebrate host, and the extracellular promastigote develops within the insect vector. The female phlebotomine sand fly becomes infected by ingesting amastigotes during a blood meal. In the sand fly gut the amas-tigotes differentiate into infectious metacyclic promastigotes that infect the vertebrate host during the next blood meal. The surface lipophos-phoglycan (LPG) plays a central role in the parasite’s entry and survival in host cells. Immunomodulatory factors present in the sand fly saliva may enhance the infectivity of the parasite.11 Once introduced into the skin the promastigotes are phagocytosed (through complement–complement receptor-mediated coiling phagocytosis) by neutrophils, dendritic cells, and macrophages, where they transform to amastigotes and replicate within the acidic and hostile environment of the phagolys-osome. Eventually the macrophages rupture and release amastigotes to infect other macrophages or a sand fly.

Tissue damage in the localized forms of leishmaniasis is mediated largely by an exuberant granulomatous response. In the case of localized cutaneous leishmaniasis (LCL) this response mediates parasite killing, so in most cases the tissue damage is short-lived and results in only a local scar. The extensive tissue destruction seen in mucosal leishmaniasis (ML) is mediated by a hyperreactive cellular immune response characterized by high levels of serum TNF-α and strong antigen-specific Th1 cytokine responses. An allelic association between ML and two different polymor-phisms in the TNF-α and TNF-β genes has been described. Patients with diffuse cutaneous (DCL) and visceral leishmaniasis (VL) are aner-gic to Leishmania antigens, and the infection is relentlessly progressive.

INNATE IMMUNITY

Much of what we know of immunity in leishmaniasis comes from stud-ies of inbred mouse strains, which demonstrate a genetically determined spectrum of innate and adaptive immune responses that determine the outcome of infection. The innate immune response to Leishmania is mediated by NK cells, cytokines and phagocytes. The production of IL-12 early in the course of infection, by dendritic cells but probably not by macrophages, leads to the early activation of NK cells and the produc-tion of IFN-γ. Chemokines (IP-10, MCP-1 and lymphotactin), as well as LPG-activated TLR4 on NK cells, can also promote early NK cell activation. Activated NK cells have been shown to be cytolytic for Leishmania-infected macrophages, but NK cell-derived IFN-γ plays a more prominent role in host defense by activating macrophages to kill the intracellular parasite through the generation of reactive oxygen inter-mediates (ROI) or reactive nitrogen intermediates (RNI). Parasite-induced MyD88-dependent signaling through TLR2, TLR3, and TLR4 contributes to macrophage activation and NO production. Activated polymorphonuclear leukocytes kill parasites primarily through oxidative mechanisms. IFN-α and IFN-β participate in the early induction of NO and control of parasite replication early in infection. The production of NO has a critical positive feedback effect on the IL-12-mediated NK-cell response.

ACQUIRED IMMUNITY

In an endemic area the prevalence of DTH skin test positivity increases and the incidence of clinical disease decreases with age, indicating the acquisition of immunity in the population over time. Retrospective epi-demiological studies indicate that most individuals with prior (primary) infection (subclinical or healed) are immune to a subsequent clinical infection. Following primary infection parasites persist for the life of the host. In the murine model the persistence of parasites is required for the maintenance of long-term immunity.

There is extensive evidence from experimental models that cellular immune mechanisms mediate acquired resistance to Leishmania infec-tion, and human studies have generally confirmed this.12 Anti- leishmanial antibodies, which are produced at a low level in LCL and at a very high level in VL, play no role in protection. A high antibody level is a marker of progressive disease in VL, and in mice antibodies can promote IL-10 production and the maintenance of a Th2 response.13 Although there are some differences among the different Leishmania species, the general mechanisms of cellular immunity can be summarized (Fig. 28.2). Following infection in the skin, Langerhans’ cells phagocy-tose and transport Leishmania to the regional lymph node, where they induce a T-cell response. Acquired immunity in murine cutaneous leish-maniasis caused by L. major is mediated by parasite-induced production of IFN-γ by CD4 T cells (Th1 subset). CD4 T cells are absolutely required, but immunity to cutaneous disease can develop in the absence of CD8 T cells. Both CD4 and CD8 T cells are required for an effective defense against murine visceral L. donovani infection, but the precise role of CD8 T cells is unclear. Generation of the Th1 response is critically dependent on CD40/CD40L-mediated IL-12 production and is driven by NK cell-derived IFN-γ. IL-27 plays a transient role in the develop-ment the Th1 response to L. major in some models, probably by counter-ing the effects of early IL-4 production.14 IL-12 is required for the maintenance of immunity. Depletion of IL-12, or disruption of the

LE

ISH

MA

NIA

SP

P.

INFECTION AND IMMUNITY

438

28

IL-12 gene, IL-18 gene or STAT4 gene (critical to IL-12 signaling), subverts Th1 cell development and renders resistant mice susceptible. Tumor necrosis factor-α (TNF-α) contributes to protective immunity by synergizing with IFN-γ to activate macrophages. Recently, NF-κB fam-ily members have been shown to regulate T-cell responses and immunity to L. major infection in mice.

The generation of RNI by activated macrophages is the primary mechanism of parasite killing in the murine model.15 The Fas/Fas ligand pathway of CD4 T cell-induced apoptosis is also involved in the elimina-tion of parasites. Although IFN-γ-induced production of NO may not be detectable in human macrophages, inhibition of nitric oxide synthase 2 (NOS2) was shown to impair killing of intracellular Leishmania.

Studies in the murine model of L. major infection have led to the idea that two populations of CD4 T cells mediate immunity induced by pri-mary infection.16 Effector memory cells, which are short-lived and dependent on the persistence of antigen, respond rapidly to secondary infection by migrating to the infected tissue and generating effector cytokines. Central memory T cells, which can be maintained in the

absence of persistent antigen, circulate throughout the lymphatic system and upon secondary challenge migrate to and proliferate in the draining lymph node, gain the capacity to produce IFN-γ, and then migrate to the site of infection. Thus, central memory T cells act as a reserve of antigen-reactive T cells that can expand and become effector T cells in response to secondary antigenic challenge. There are a number of adaptive immune mechanisms that promote parasite replication and disease.17 The progression of murine L. major infection has been correlated with the expansion of Th2 cells and the production of IL-4, IL-5 and IL-10. In susceptible mice, IL-4 production within the first day of infection was shown to downregulate IL-12 receptor β-chain expression and drive the response to a Th2 phenotype. However, other nonsusceptible mouse strains appear to be able to overcome an early IL-4 response and develop a resistant phenotype, and susceptibility to some L. major strains is not strictly mediated by IL-4 (IL-13 may contribute). Additional factors, such as IL-10, TGF-β, or PGE2, are required to maintain the Th2 response and the susceptible phenotype.16 Recent evidence indicates that susceptibility in the BALB/c mouse model of L. major infection is as much a result of an inherent defect in the Th1 development pathway and IL-10 production as it is related to the effects of IL-417. Besides inhibi-tion of IFN-γ production, these factors suppress macrophage activation. The antigen-specific unresponsiveness of CD4 T cells during active VL is mediated in part by the engagement of the negative co-stimulatory receptor CTLA-4.

Peripheral blood mononuclear cells (PBMCs) isolated from patients with localized or subclinical leishmaniasis demonstrate a Th1 response to Leishmania antigens, and in general the intralesional cytokine profile is one of a dominant Th1 response. Patients with ML exhibit vigorous T-cell responses; it is postulated that this hyperresponsive state contributes to the prominent tissue destruction of ML. Patients with DCL resemble the progressive infection caused by L. major in BALB/c mice. Such patients demonstrate minimal or absent Leishmania-specific lymphopro-liferative responses, and the Th2 cytokine mRNAs were prominently expressed in DCL lesions. During active VL in humans there is a marked depression of Leishmania-specific lymphoproliferative and IFN-γ responses, as well as an absence of DTH response to parasite antigens. This anergy appears to be mediated, at least in part, by a suppressive effect of IL-10 and low levels of IL-12. Successful treatment of active disease restores an antigen-specific Th1 response.

EVASION OF HOST IMMUNITY

The Leishmania parasite has numerous ways in which it adapts to and survives within the vertebrate host.15, 17 In the skin the promastigotes are preferentially phagocytosed by macrophages that, unlike dendritic cells, do not actively participate in T-cell priming. The parasite’s surface LPG (and to a lesser extent the surface protein gp63) plays an important role in the entry and survival of Leishmania in the mammalian host by confer-ring complement resistance, and by facilitating the entry of complement-opsonized parasites into the macrophage without triggering a respiratory burst. Macrophage phagosome–endosome fusion and phagolysosomal biogenesis are also inhibited by the parasite LPG.

Leishmania-infected macrophages have a diminished capacity to initi-ate and respond to a T-cell response, and the impaired antimicrobial effector activity provides a safe haven for the intracellular parasite.15 Infected macrophages have decreased synthesis of cytokines (IL-1, IL-12) and blunted IFN-γ-mediated activation (reduced MHC class II and

DC

Leishmania promastigotes

IL-10TGF-PGE2

TNF-IL-10TGF- IL-10

TGF-

IL-12

IL-12IFN-

IFN-

IFN-

IFN-

IFN-

IFN-

IL-4IL-10

IL-4IL-10Th2

NK

Th1

Parasitereplication

Deactivation

Macrophage

Parasite killing

RNIROI

Activation

Fig. 28.2 Immunity in leishmaniasis. Exposure of dendritic cells to

parasites or parasite antigens leads to the release of IL-12, which

induces NK cells to produce IFN-γ and drives the acquired immune

response toward a protective Th1 phenotype. IL-12 production by

dendritic cells and IFN-γ production by NK and Th1 cells negatively

regulates the Th2 response. IFN-γ activates macrophages to kill the

intracellular pathogen. In genetically susceptible individuals a counter-

regulatory Th2 cytokine response can suppress the Th1 response and

deactivate infected macrophages, leading to parasite replication and

uncontrolled infection. Counterprotective macrophage-derived

cytokines can also inhibit the Th1 response, stimulate the Th2

response, and deactivate the macrophage through an autocrine loop.

Activating stimuli are shown by solid arrows, and deactivating stimuli

are shown by dashed arrows.

LE

ISH

MA

NIA

SP

P.

Immune responses to protozoans 28

439

co-stimulatory molecule expression, and reduced production of ROI and RNI) through the disruption of signal transduction pathways involving JAK/STAT, protein kinase C, p38 MAPK, ERK, AP-1, and NF-κB. Signaling mediated by tyrosine phosphorylation is reduced by the rapid induction of the host phosphotyrosine phosphatase SHP-1. Conversely, there is increased synthesis of the immunosuppressive molecules IL-10, TGF-β and prostaglandin E2. Together these contribute to impaired antigen presentation to T cells. The IL-4/IL-13-enhanced expression of arginase by infected macrophages (termed alternatively activated macro-phages) contributes to depletion of -arginine and reduced NO produc-tion. Recently, IL-10-producing CD4+CD25+ T-regulatory cells at the site of chronic infection have been shown to play a role in the mainte-nance or progression of infection.18 IL-10 has an essential role in parasite persistence, as mice with IL-10 deletion or IL-10R blockade are able to eliminate the parasite.19

� TRYPANOSOMA CRUZI �PATHOGENESIS

Trypanosoma cruzi is transmitted to the mammalian host when the infectious metacyclic trypomastigote, which is deposited on the skin in the feces of the reduvid insect vector while it takes a blood meal, is scratched into the wound or transferred to a mucous membrane (e.g., the eyes). The trypomastigotes can infect almost any cell type, and replicate as amastigotes in the cytoplasm. Eventually the amastigotes transform back into trypomastigotes and rupture the cell to enter the bloodstream, whence they invade other cells or are picked up by another insect vector.

Following primary infection the parasites replicate locally and then disseminate through the bloodstream to a variety of tissues. Muscle and glial cells are the most frequently infected, and acute myocarditis or meningoencephalitis can develop. In most cases, however, primary infec-tion occurs without clinical symptoms and the infected individual may enter an indeterminate phase of asymptomatic seropositivity. Only 10–30% of chronically infected individuals will ultimately develop sympto-matic Chagas’ disease, usually involving the heart or gastrointestinal tract. Pathologically there are few parasites observed in cardiac tissue, but an intense chronic inflammatory infiltrate with fibrosis and loss of mus-cle fibers is evident. In the digestive tract there is lymphohistiocytic infiltration of the myenteric plexuses, with a dramatic reduction in the number of ganglion cells.

The tissue damage of acute T. cruzi infection is the result of a direct effect of the parasite and the acute host inflammatory response. In chronic infection the balance between immune-mediated parasite containment and host-damaging inflammation determines the course of disease. The pathological mechanisms related to chronic Chagas’ disease are controversial, but whether tissue damage is caused directly by parasites or indirectly through parasite-directed inflammatory or autoimmune mechanisms, it is clear that parasite persistence is required for disease.20, 21 Autoimmunity could arise from molecular self-mimicry by parasite antigens, or by the release of self-molecules from damaged or dying host cells within the environment of an acti-vated innate immune response. The production of IL-10 by T. cruzi-infected cells may downregulate the pathologic cellular immune response.22

INNATE IMMUNITY

The early innate immune response to T. cruzi infection is mediated pri-marily by NK cells and macrophages.20 Macrophages and DCs exposed to T. cruzi trypomastigote antigens produce proinflammatory cytokines, including IL-12 and TNF-α, through a MyD88-dependent mechanism. MyD88-deficient mice had impaired inflammatory responses and host defense against T. cruzi. IL-12 activates NK cells to secrete IFN-γ, which synergizes with TNF-α to activate macrophages to control parasite rep-lication. The generation of NO is the primary trypanocidal mechanism in murine macrophages. A number of trypomastigote antigens, including free GPI anchors, glycoinositol phospholipids (GIPLs), GPI-linked glycoproteins, and GPI-mucins activate the innate immune response, at least partly through TLR-2.

ACQUIRED IMMUNITY

In infected mice the parasitemia (trypomastigotes) increases until 3–4 weeks after infection, when either the mice die or the infection is control-led by the acquired immune response. Antibodies contribute to immunity through opsonization, complement activation and antibody-dependent cellular cytotoxicity.

Several lines of evidence establish the importance of T cells in acquired immunity to T. cruzi infection. Parasite-specific CD4 and CD8 T cells are activated in response to infection, and mice lacking CD4 or CD8 T cells have impaired ability to control the infection. CD8 T cells with cytotoxic activity against T. cruzi-infected cells have been identified in infected mice, and these cells confer protection against challenge when passively trans-ferred to naive mice. In the early stage of infection CD4 T cells are the predominant subset recruited to the myocardium, but activated CD8 T cells soon dominate the inflammatory process in cardiac tissue. Cytokine production (IFN-γ and TNF-α) by parasite-specific CD8 T cells is more important than cytolytic activity in the control of infection.23

T. cruzi infection leads to a mixed Th1/Th2 cytokine response, and in general the Th1/Th2 balance determines resistance or susceptibility. As noted earlier, IFN-γ, produced in early infection by NK cells and later by T cells, clearly has an important protective function. Its protective effect is dependent on IL-12, as neutralization of IL-12 or deletion of STAT4 leads to increased parasitemia and earlier death, and the protection afforded to mice by the administration of IL-12 was abrogated by neu-tralization of IFN-γ and TNF-α. IL-4 does not appear to play a major role in susceptibility to T. cruzi infection, but IL-10 promotes parasite replication by inhibiting macrophage trypanocidal activity. IL-10 also plays a critical role in minimizing inflammation-mediated tissue pathol-ogy by regulating the Th1 and proinflammatory cytokine (predomi-nantly TNF) responses.22 Similarly, TGF-β has been shown to inhibit macrophage trypanocidal activity and increase parasitemia and mortality. In addition to the production of these regulatory cytokines, the secretion of prostaglandins and NO, the induction of apoptosis of T and B cells, and the expansion of a myeloid suppressor cell population serve to con-trol the intensity of the immune response.

EVASION OF HOST IMMUNITY

A significant part of the pathogenesis following T. cruzi infection is its dis-semination through the bloodstream to many tissues. T. cruzi bloodstream trypomastigotes resist complement-mediated lysis because the parasite has

TR

YP

AN

OS

OM

A C

RU

ZI

INFECTION AND IMMUNITY

440

28

a complement regulatory protein (GP-160), which is functionally similar to mammalian decay-accelerating factor in that it inhibits C3 convertase formation and activation of the alternate complement pathway.

The establishment of chronic infection by T. cruzi is favored by a gener-alized depression of T-cell responses.24 A number of different mechanisms may contribute to this, including low IL-2 production or IL-2 receptor expression; downregulation of components of the T-cell–receptor com-plex; T-cell receptor dysfunction; apoptosis of T cells; defects in the processing and presenting of antigens in the class II (but not the class I) pathway; T-cell or macrophage suppressor activity; and prostaglandin E2 production.25 Within foci of myocarditis, apoptosis of both parasites and host cells occurs; the phagocytosis of these apoptoic cells by macro-phages leads to their deactivation or acquisition of an alternatively acti-vated phenotype. Deactivated or alternatively activated macrophages may serve as an important host cell for parasite replication and persistence. The rapid escape of the parasite from the phagosome into the cytoplasm through the action of acid-activated porins enables the organism to avoid enzymatic destruction.

� TOXOPLASMA GONDII �PATHOGENESIS

Transmission occurs via the ingestion of oocysts, which are shed in the feces of felines or present in undercooked meat. Following the oral ingestion of cysts, intestinal epithelial cells are infected by trophozoites that enter via the laminin receptor. The parasite transforms into intra-cellular tachyzoites that replicate within a parasitophorous vacuole and

ultimately rupture the cell. The released tachyzoites can invade virtually any nucleated cell type and disseminate to all tissues. Under pressure from the host immune response, tachyzoite replication is controlled and tissue cysts, which are a modified parasitophorous vacuole containing slowly replicating bradyzoites, are formed. The tissue cysts persist as a chronic latent infection as long as the host immune function is intact. The outcome of the host–parasite interaction is determined by the deli-cate balance between proinflammatory antimicrobial mechanisms and anti-inflammatory mechanisms that restrict tissue damage. If the latently infected person is immunosuppressed reactivation occurs, and tachyzoites are released to infect more cells. Because tissue cysts (bradyzoites) are found in proportionately larger numbers in the brain, reactivation of latent infection in the immunocompromised host is most commonly manifest as encephalitis.

INNATE IMMUNITY

Infection with T. gondii induces IL-12-dependent activation of NK cells. Activated NK cells produce IFN-γ, which in turn activates macro-phages to limit parasite replication. Macrophage and dendritic cell production of IL-12 is enhanced by NK cell-derived IFN-γ. IL-12 production, which is essential to host resistance to T. gondii, is triggered through a MyD88-dependent mechanism, at least in part through the interaction of parasite-derived proteins and TLR1126 and activation of CCR5. IL-12, IFN-γ, TNF-α and NK cells contribute to the control of the early stages of T. gondii infection in both immunocompetent mice and SCID mice (lacking T and B cells). The effects of IL-12 are augmented by other macrophage products, such as TNF-α, IL-1 and

EVIDENCE FOR AUTOIMMUNE AND PARASITE-INDUCED INFLAMMATORY MECHANISMS

IN CHRONIC CHAGAS’ DISEASE

Evidence for autoimmune-mediated disease Evidence for parasite-induced inflammatory disease

Inflammatory disease is present in tissues with few

or no parasites seen on routine histopathology studies.

Sensitive parasite detection techniques (PCR,

immunohistochemistry) show a strong correlation between the

presence of parasites (or parasite material) and the severity of

inflammatory disease.

There is a peculiar pattern of organ involvement (heart and

gastrointestinal tract) in patients with chronic disease.

Organs free of parasites (by sensitive parasite detection techniques)

are also free of disease.

There is a long delay in the onset of chronic disease following

infection, and only a minority of infected persons will ever

develop disease.

The absence of an effective cellular immune response (in mice or

humans) almost invariably exacerbates rather than reduces the

parasite burden and disease.

There is wide variability in the expression of disease among infected

people.

In chronically infected mice the destruction of a transplanted heart is

dependent on parasite infiltrating the transplanted tissue.

Self-reactive antibodies and T cells have been demonstrated in

infected people and in experimental animals. The level of antibodies

to the ribosomal P protein (R13 peptide) and cardiac myosin (B13

antigen) correlate with cardiac disease.

The degree of disease in hearts transplanted into chronically infected

mice correlates with the level of parasite burden in the transplanted

tissue.

Transient or limited disease has been reported in experimental

models following lymphocyte transfer.

Reduction of the parasite burden by chemotherapy leads to

decreased tissue inflammation and disease.

CLINICAL RELEVANCE

TO

XO

PL

AS

MA

GO

ND

II

Immune responses to protozoans 28

441

IL-15. The early proliferation of NK cells is dependent on CD4 T-cell production of IL-2.

T cells expressing the αβ T-cell receptor are also a source of IFN-γ early in the course of infection. In mice there is evidence that naïve T cells are activated through a superantigen-like effect, but the T. gondii-induced activation of T cells from previously unexposed human donors is dependent on the classic pathway of antigen processing and presenta-tion (Chapter 6). T cells expressing the γδ receptor are also an early source of IFN-γ and TNF-α, leading to macrophage activation and kill-ing of T. gondii. γδ T cells also induce macrophage heat-shock protein, which may prevent infected macrophages from undergoing apoptosis. In murine toxoplasmosis the virulence of T. gondii correlates with the abil-ity of the parasite to induce macrophage apoptosis.

The mechanism(s) of IFN-γ-mediated macrophage activation for the control of early parasite replication is unclear. The production of NO by activated macrophages does not appear to play a significant role in the early stage of infection, but it is important in chronic infection. Other possible IFN-γ-dependent mechanisms that contain parasite replication include the generation of reactive oxygen intermediates and the limita-tion of intracellular tryptophan.

ACQUIRED IMMUNITY

The systemic antibody response does not play a role in acquired immu-nity to T. gondii. Mucosal IgA, however, does play a role in resistance to oral infection with T. gondii cysts. T. gondii is a potent activator of CD4 and CD8 T cells. The importance of T cells in acquired immunity to T. gondii is demonstrated by the following observations: 27 (1) athymic nude mice are highly susceptible to both virulent and avirulent strains of T. gondii; (2) depletion of T cells in chronically infected mice results in the reactivation of infection and death; (3) adoptive transfer of immune T cells confers protection on naïve mice against challenge with virulent parasites; (4) MHC class I and class II antigens have substantial influ-ence on host resistance and susceptibility; and (5) patients with defects in T cell-mediated immune responses (e.g., patients with AIDS) are at risk for reactivation of latent infection.

NK cell-derived IFN-γ drives the differentiation of T cells into type 1 CD4 and CD8 T cells that are essential to acquired immunity. The route by which peptides enter the class I and class II antigen-processing pathways have not been fully defined, but evidence sup-ports exogenous soluble antigens, perhaps derived from dead or dying parasites, as the source. Parasite-specific cytolytic T cells have been demonstrated in both murine and human infection. CD8 T cells are capable of transferring protection to naïve mice, primarily through the generation of IFN-γ; the cytolytic activity of such cells is a secondary mechanism.27 CD4 and CD8 T cells act synergistically to prevent cyst reactivation during chronic infection, and there is evidence that some human parasite-specific CD4 T cells have cytolytic activity. IL-27 negatively regulates T cell activation by a STAT1-dependent mechansism.14

Macrophage activation is the key effector mechanism in acquired immunity to T. gondii. In the case of murine toxoplasmosis, IFN-γ and TNF-α induce NO synthase and the production of reactive nitrogen intermediates. Neutralization of these cytokines leads to decreased expression of NOS2 and reactivation of chronic infection. As previously noted, the role of reactive nitrogen intermediates in the effector function of human macrophages is uncertain. Several other effector mechanisms,

including IFN-γ-mediated degradation of tryptophan, the generation of reactive oxygen intermediates and the production of leukotrienes, have been implicated in the control of T. gondii in human macrophages. Anti-inflammatory molecules, particularly IL-10, play an important role in modulating the adaptive immune response and restricting host tissue damage.28 IL-10-deficient mice develop uncontrolled inflammation and tissue necrosis, which are mediated by overproduction of IFN-γ and TNF, leading to death.

EVASION OF HOST IMMUNITY

T. gondii escapes early macrophage killing in a number of ways.28 Virulent parasites are protected by localization to the parasitophorous vacuole that does not fuse with host cell lysosomes, probably because the PV membrane proteins are of parasite rather than host origin and the vacuole is not acidified. The infected macrophage is also a subop-timal target for T cell-induced immunity because of reduced expres-sion of MHC class II and co-stimulatory molecules. Infection also induces the production of counter-regulatory molecules, such as the cytokines IL-10 and TGF-β, an eicosanoid lipoxin A4, or members of the suppressor of cytokine synthesis (SOCS) family. These protect the host by downregulating a potentially pathologic inflammatory response, but also inhibit Th1 cytokine synthesis and macrophage antimicrobial activity. The production of oxide-reducing enzymes by the intracellular parasite can also neutralize effector molecules such as NO or superoxide.

� ENTAMOEBA HISTOLYTICA �PATHOGENESIS

E. histolytica cysts are ingested through the consumption of food or water contaminated by feces. After excystation, the motile trophozoites pene-trate the mucous barrier by mechanical disruption. The trophozoites adhere to the colonic epithelial cells by a galactose/N-acetylgalactos-amine-inhibitable adhesin (Gal/GalNAc lectin). Epithelial cells with adherent amebae undergo shortening of microvilli and apical separation, and the trophozoites penetrate between the epithelial cells. This invasion is facilitated by cysteine proteases and a surface metallocollagenase, which attack the components of connective tissue ground substance causing ulceration of the colonic mucosa and submucosa (Fig. 28.3). The trophozoites of E. histolytica can lyze multiple cell types, including neu-trophils, which release enzymes that further damage the tissue. The cytotoxic effects of amebae are mediated by a secreted cysteine protease, the Gal/GalNAc lectin, phospholipase A, and contact-dependent cytoly-sis, in which an ion channel (amebapore) is inserted into the membrane of target cells. E. histolytica can induce apoptosis in mammalian cells by a caspase-dependent, TNF-α/Fas-independent process. Amebic liver abscesses develop when trophozoites erode through the intestinal sub-mucosa, enter the portal circulation and are deposited in the liver.29, 30

INNATE IMMUNITY

It is now thought that innate immunity is the key factor in the contain-ment and resolution of amebic colitis.30, 31 Trophozoites that successfully breach the protective mucous barrier bind to epithelial cells, stimulating the secretion of pre-IL-1β, IL-1α, IL-6, IL-8, GRO-α and GM-CSF,

EN

TA

MO

EB

A H

IST

OLY

TIC

A

INFECTION AND IMMUNITY

442

28

which attract leukocytes to the site of invasion.32 Neutrophils are the most important effector cells in clearing E. histolytica infection.31 Studies in IFN-γ receptor gene knockout mice suggest a role for IFN-γ in innate immunity against E. histolytica.30

ACQUIRED IMMUNITY

Invasive amebiasis produces a humoral immune response in most patients, but the protection afforded is incomplete, i.e., an amebic liver abscess may progress despite an exuberant host antibody response. Nevertheless, anti-bodies protect against intestinal colonization and reinfection.4 The exact role of IgA in host defense against amebiasis is unclear; IgA deficiency does not cause more frequent or severe disease.33

The role of cell-mediated immunity in the control of amebiasis is also not fully defined. Dissemination of infection occurs in some condi-tions where cell-mediated immunity is impaired,4, 33 but the AIDS epidemic has not resulted in an increase in cases of severe amebiasis. In rodents immunized with the Gal/GalNac lectin, or in humans who have antilectin antibodies, T lymphocytes produce IL-2 and IFN-γ when stimulated with one of the lectin’s subunits. The Gal/GalNac lectin also triggers IFN-γ-primed macrophages to produce the

amebicidal molecule NO. Early amebic liver abscess in gerbils is associ-ated with a mixed Th1/Th2 response, but with chronic infection the gerbils mount a Th1-like response. Gerbils cured of amebic liver abscess by antibiotic therapy likewise have a Th1 immune response and are resistant to reinfection.

EVASION OF HOST IMMUNITY

E. histolytica utilizes a number of strategies to circumvent the immune defenses of the host. It resists complement-mediated lysis during hema-togenous spread by proteolytic degradation of C3 and C5. In addition, the Gal/GalNac lectin binds to C8 and C9, preventing assembly of the C5b-9 membrane attack complex 4 (Chapter 20).

The cytolytic capability of E. histolytica affords protection from neu-trophils, macrophages and eosinophils unless these cells are activated.33 Cytolysis by E. histolytica may occur via necrosis and apoptosis; the use of the host’s apoptotic machinery abrogates the local inflammatory response. Trophozoites also inhibit the macrophage respiratory burst and the pro-duction of IL-1 and TNF-α. A protective antibody response is subverted by the degradation of IgA and IgG by amebic cysteine proteases, and by capping, ingesting or shedding ameba-specific antibodies.33

Pathogen Intestinal responseInnate immune response

ComplementMacrophage

NeutrophilEosinophilNK cell

Enterocyte(Cryptosporidium)

Inflammatorymediators

Villous atrophy andcrypt hyperplasia

Epithelialdamage

Erosions andulcerations

IL-6, IL-8, IL-1GM-CSF, GRO ,prostaglandinsROI, RNIProteases

Trophozoite

Trophozoite

Sporozoite

Cytokines

B- and T-lymphocyte activation SecretionMalabsorption

ExudationAcquired immune response

Host protection

Giardia

Cryptosporidium

Entamoeba

DiarrheaFig. 28.3 Immunopathogenesis of intestinal protozoal pathogens. After adherence (Giardia and Entamoeba) or epithelial invasion (Entamoeba

and Cryptosporidium), there is release of various inflammatory mediators from macrophages and neutrophils. This causes the activation of

resident phagocytes and recruitment of phagocytes into the lamina propria. Enterocyte death may be due to direct action of the parasites or to

immune-mediated damage from complement, cytotoxic lymphocytes, proteases, and reactive oxygen and nitrogen intermediates (ROI and RNI,

respectively). The inflammatory mediators also act on enterocytes and the enteric nervous system, inducing the secretion of water and chloride.

Under the influence of activated T lymphocytes the response to enterocyte damage is that the crypts undergo hyperplasia and the villi become

shorter (villous atrophy). The immature hyperplastic cells have poor absorptive ability, but retain secretory ability. Damage to the epithelium can

cause leakage (exudation) from lymphatics and capillaries. Similar mechanisms are probably responsible for the diarrhea that occurs in infection

with Cyclospora and Isospora. Isospora is unique in causing an eosinophilic infiltrate.

EN

TA

MO

EB

A H

IST

OLY

TIC

A

Immune responses to protozoans 28

443

Amebic antigens may cause selective activation of Th2-type T cells, causing macrophage deactivation and anergy. 4 Macrophages exposed to amebic lysates secrete prostaglandin E2, which interferes with MHC II expression and TNF-α production. Patients with amebic liver abscesses have decreased CD4/CD8 T-cell ratios and decreased T-cell prolifera-tion, which may be related to reduced IL-2 production.

� GIARDIA LAMBLIA �PATHOGENESIS

After ingestion of the Giardia cyst in fecally contaminated food or water, excystation occurs, with the release of trophozoites. The tro-phozoites multiply in the small bowel. As they transit through the lower intestine they encyst, allowing the organism to survive in the environment and to be transferred to another host. The Giardia trophozoite initially adheres to intestinal epithelium via a surface mannose-binding lectin. Histopathologic changes in symptomatic giardiasis range from a normal appearance to villous atrophy, crypt hyperplasia, epithelial damage, and chronic inflammatory infiltrate in the lamina propria (Fig. 28.3). The factors responsible for the struc-tural changes in the small bowel are not well defined, but may include injury from adherence, and the release of cytotoxins, including pro-teases. Furthermore, epithelial damage may be mediated by the host cellular immune response.34

INNATE IMMUNITY

Giardia has a limited capacity to neutralize reactive oxygen species, which are produced by intestinal epithelial cells. The antimicrobial peptide defensin is produced by Paneth cells and thus may also participate in host defense against Giardia. Few macrophages are found in the intestinal mucosa during giardiasis, which suggests they do not play an important role in the innate immune response.35 The infection is rare in breastfed infants because breast milk contains free fatty acids lethal to Giardia cysts and, in endemic areas, anti-Giardia antibodies.34

ACQUIRED IMMUNITY

Several lines of evidence suggest the importance of the humoral immune response in the control of giardiasis.35 Infection with Giardia results in the production of anti-Giardia antibodies in the serum and mucosal secretions. Human immunodeficiency syndromes that affect antibody production are associated with chronic giardiasis.

There is evidence for a role of T cell-dependent immunity in the con-trol of giardiasis, but the mechanisms of immunity have not been fully defined.35 T cell-deficient mice and mice treated with anti-CD4 anti-body are unable to control Giardia infection. CD8 T lymphocytes are not important in protective immunity.

EVASION OF HOST IMMUNITY

Giardia undergoes surface antigenic variation by altering a group of variant-specific surface proteins (VSPs). Selection occurs by an immune-mediated process, because switching occurs when intestinal anti-VSP IgA responses are first detected.36 Giardia lamblia also produces a pro-tease that cleaves IgA.

� CRYPTOSPORIDIUM PARVUM �There are three intestinal coccidian parasites of humans that are intracel-lular parasites of enterocytes: Cryptosporidium parvum, Isospora belli and Cyclospora cayetanensis. Cryptosporidium has the greatest epidemiologic significance: in 1993 a huge outbreak involving 403 000 persons occurred in Milwaukee, Wisconsin.37 Because of their similarity, only the immu-nology of cryptosporidiosis will be discussed in detail.

PATHOGENESIS

Infection with Cryptosporidium occurs when sporulated oocysts are ingested and excyst in the proximal small bowel, invade the intestinal epithelial cells (facilitated by a cysteine protease), develop into tropho-zoites and undergo schizogony, with a resultant merozoite-containing schizont. The merozoites are extruded and invade other epithelial cells. The merozoites may continue an asexual cycle or develop into macro- or microgametes that fuse to form oocysts.

Histologically, in the infected small bowel there is villous atrophy and blunting, and crypt hyperplasia with increased infiltration of lym-phocytes, macrophages and plasma cells. Intraepithelial lymphocytes are uncommon; neutrophils and occasional eosinophils are present between the epithelium and the lamina propria. Disorganized cells undergoing necrosis replace normal enterocyte architecture (Fig. 28.3). There is an association between the degree of intestinal injury and malabsorption and the intensity of infection, as measured by oocyst excretion.37

INNATE IMMUNITY

Knowledge of the immune response to cryptosporidiosis has been ham-pered owing to the lack of experimental models of infection. Acute infec-tion in an immunocompetent mouse older than 3 weeks is difficult to establish, so the neonatal mouse model has been most commonly studied. The increased susceptibility of neonates (including humans) may be related to an undeveloped IL-12-dependent IFN-γ pathway.

Because of the parasite’s intracellular location near the luminal sur-face of the enterocyte, the macrophages of the lamina propria are spa-tially isolated from the parasite. Thus, the intestinal epithelium mounts its own assault on the invading microbe by the activation of NF-κB and the release of TNF-α and the chemokines IL-8, RANTES, and GRO-α, which act as chemoattractants and activators of neutrophils.38 TNF-α also inhibits parasite replication. Infected epithelial cells may attempt to eliminate the parasite through apoptosis;38 however, NF-κB activation protects the infected epithelial cells from apoptosis, thereby facilitating parasite survival and replication.39

Infected intestinal cells also release TGF-β, which decreases necrosis and stimulates the synthesis of extracellular matrix proteins, which limit epithelial damage.38 Antimicrobial peptides (the defensins) released by intestinal cells also have anti-cryptosporidial activity. There are contra-dictory data on the role of NO in the control of cryptosporidosis.38

ACQUIRED IMMUNITY

The relative contribution of antibody responses to the control of crypt-osporidosis is uncertain.39 The enterocyte membrane enveloping the parasitophorous vacuole contains C. parvum antigens that are recognized

CR

YP

TO

SP

OR

IDIU

M P

AR

VU

M

INFECTION AND IMMUNITY

444

28

by antibody. Serum and mucosal antibody responses accompany resolu-tion of the diarrhea and oocyst shedding, and may contribute to resistance to, or decreased severity of, reinfection.

CD4 T cells play a central role in the control of cryptosporidiosis. Mice that lack CD4 T cells experience persistent infection. In patients with AIDS, severe disease or biliary involvement (an indicator of chronic infection) is usually seen in those with CD4 counts <50/mm3. HIV-infected patients with CD4 counts >200 usually experience only transient disease. In contrast, peripheral CD8 cells have only a minor role in the immune response. Adoptive transfer of intraepithelial lym-phocytes (IELs) from C. muris-immune BALB/c mice results in para-site clearance in SCID mice.39 In immunocompetent mice CD4 IELs increase and initiate early control of infection, whereas cytotoxic CD8 IELs appear later and function in parasite elimination.40 In murine models, both CD4 cells and IFN-γ of T- and non-T-cell origin are necessary to prevent or limit the severity of infection. In IFN-γ-defi-cient mice infection with C. parvum leads to mucosal destruction, rapidly progressive wasting and death. However, in humans, IFN-γ may not play a major role in controlling primary infection, but may be more relevant to the control of infection in previously exposed individuals.39

Recently, a role for γδ T cells in host defense against C. parvum has been recognized. These cells are rapidly recruited to the site of crypt-osporidial infection, and deficiency leads to increased susceptibility to infection in mice. The role of γδ T cells in the human immune response in cryptosporidosis is unknown.38 IL-15 may be involved in the control of primary infection in naïve human hosts by the activation of γδ T cells, NK cells, and perhaps enterocytes.39

The role of CD154–CD40 interaction in the control of C. parvum infection has recently been elucidated. CD154 is a TNF-like molecule expressed on the surface of activated CD4 cells. CD154 binds to CD40, which is expressed on B cells, macrophages, dendritic cells, epithelial cells, and T cells. The interaction stimulates the production of IL-12, IFN-γ, and NO, and induces apoptosis in infected cells. Knockout of these molecules results in persistent infection.39

� TRICHOMONAS VAGINALIS �PATHOGENESIS

Trichomonas vaginalis is a flagellated protozoan parasite of the human urogenital tract that exists only as a trophozoite. Its adherence to the vaginal squamous epithelium is facilitated by a number of adhesins. Trichomonas causes tissue damage by contact-dependent cytolysis due to pore-forming proteins and proteases, and secretion of a glycoprotein cell-detaching factor that causes sloughing of the vaginal epithelium. Levels of the cell-detaching factor correlate with the severity of the disease, and vaginal antibodies directed against this factor modulate its effects. Inflammation in the genital mucosa and submucosa leads to copious secretions, and the surface epithelium may slough, causing focal erosions and hemorrhage.

The increased risk of HIV transmission in women with trichomonia-sis may be due to increased recruitment of inflammatory cells, mucosal erosion, or degradation of secretory leukocyte protease inhibitor (SLPI) by trichomonal proteases. Lower levels of SLPI are found in the vaginal fluid of women with trichomoniasis, which can lead to increased tissue damage and HIV transmission.41

INNATE IMMUNITY

Although trichomoniasis has recently received increased attention as a risk factor for HIV transmission and obstetric complications, there is little known about the protective immune response against this organ-ism. Trichomonas secretes a factor that promotes neutrophil chemotaxis, causing profuse leukorrhea, but the oxidative microbicidal mechanisms of the neutrophils have decreased efficacy in the anaerobic vaginal environment. 42 Macrophages can destroy trichomonads in a T and B cell-independent manner.

ACQUIRED IMMUNITY

Repeated infections with T. vaginalis do not induce immunity; however, the infection is self-limiting in most cases, so there are mechanisms of host defense. T. vaginalis induces the production of antibodies in both the serum and vaginal secretions. The serum antibody response correlates with active infection, and serum, but not vaginal, IgG from infected patients displays complement-mediated lytic activity against trichomonads in culture.43

EVASION OF HOST IMMUNITY

Although T. vaginalis activates the alternative pathway of complement, the cervical mucus and menstrual blood are low in complement. Menstrual blood also supplies iron, which upregulates trichomonal adhesins and cysteine proteases, causing the degradation of comple-ment component C3 bound to the surface of the parasite. Parasite viru-lence is thus enhanced, and the symptoms are exacerbated during menses. Cysteine proteases secreted by T. vaginalis also degrade immu-noglobulins, sabotaging the antibody response. The parasite also secretes soluble antigens that act as decoys for neutralizing antibodies or cytotoxic T cells, and disguises itself by binding to host plasma pro-teins. Phenotypic variation of surface markers is also a means of anti-body evasion.43

� REFERENCES � 1. Coler R.N., Reed S.G.. Second-generation vaccines against leishmaniasis.

Trends Parasitol 2005; 21: 244–249.

2. Good M.F.. Vaccine-induced immunity to malaria parasites and the need for novel strategies. Trends Parasitol 2005; 21: 29–34.

3. Hill A.V.. Pre-erythrocytic malaria vaccines: towards greater efficacy. Nature Rev Immunol 2006; 6: 21–32.

4. Huston C.D., Petri W.A. Jr. Host–pathogen interaction in amebiasis and progress in vaccine development. Eur J Clin Microbiol Infect Dis 1998; 17: 601–614.

5. Ramasamy R.. Molecular basis for evasion of host immunity and patho-genesis in malaria. Biochim Biophys Acta 1998; 1406: 10–27.

6. Schofield L., Grau G.E.. Immunological processes in malaria pathogenesis. Nature Rev Immunol 2005; 5: 722–735.

7. Scharton-Kersten T.M., Sher A.. Role of natural killer cells in innate resist-ance to protozoan infections. Curr Opin Immunol 1997; 9: 44–51.

8. Doolan D.L., Martinez-Alier N.. Immune response to pre-erythrocytic stages of malaria parasites. Curr Mol Med 2006; 6: 169–185.

RE

FE

RE

NC

ES

Immune responses to protozoans 28

445

9. Marsh K., Kinyanjui S.. Immune effector mechanisms in malaria. Parasite Immunol 2006; 28: 51–60.

10. Hisaeda H., Maekawa Y., Iwakawa D., et al. Escape of malaria parasites from host immunity requires CD4+ CD25+ regulatory T cells. Nature Med 2004; 10: 29–30.

11. Titus R.G., Bishop J.V., Mejia J.S.. The immunomodulatory factors of arthro-pod saliva and the potential for these factors to serve as vaccine targets to prevent pathogen transmission. Parasite Immunol 2006; 28: 131–141.

12. Solbach W., Laskay T.. The host response to Leishmania infection. Adv Immunol 2000; 74: 275–317.

13. Miles S.A., Conrad S.M., Alves R.G., et al. A role for IgG immune com-plexes during infection with the intracellular pathogen Leishmania. J Exp Med 2005; 201: 747–754.

14. Hunter C.A., Villarino A., Artis D., et al. The role of IL-27 in the develop-ment of T-cell responses during parasitic infections. Immunol Rev 2004; 202: 106–114.

15. Olivier M., Gregory D.J., Forget G.. Subversion mechanisms by which Leishmania parasites can escape the host immune response: a signaling point of view. Clin Microbiol Rev 2005; 18: 293–305.

16. Scott P., Artis D., Uzonna J., et al. The development of effector and mem-ory T cells in cutaneous leishmaniasis: the implications for vaccine devel-opment. Immunol Rev 2004; 201: 318–338.

17. Sacks D., Anderson C.. Re-examination of the immunosuppressive mecha-nisms mediating non-cure of Leishmania infection in mice. Immunol Rev 2004; 201: 225–238.

18. Belkaid Y., Piccirillo C.A., Mendez S., et al. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 2002; 420: 502–507.

19. Belkaid Y., Hoffmann K.F., Mendez S., et al. The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure. J Exp Med 2001; 194: 1497–1506.

20. Golgher D., Gazzinelli R.T.. Innate and acquired immunity in the patho-

genesis of Chagas’ disease. Autoimmunity 2004; 37: 399–409.

21. Tarleton R.L., Zhang L.. Chagas’ disease etiology: autoimmunity or parasite persistence?. Parasitol Today 1999; 15: 94–99.

22. Hunter C.A., Ellis-Neyes L.A., Slifer T., et al. IL-10 is required to prevent immune hyperactivity during infection with Trypanosoma cruzi. J Immunol 1997; 158: 3311–3316.

23. Martin D., Tarleton R.. Generation, specificity, and function of CD8+ T cells in Trypanosoma cruzi infection. Immunol Rev 2004; 201: 304–317.

24. DosReis G.A., Freire-de-Lima C.G., Nunes M.P., et al. The importance of aberrant T-cell responses in Chagas disease. Trends Parasitol 2005; 21: 237–243.

25. Bogdan C., Rollinghoff M.. How do protozoan parasites survive inside macrophages?. Parasitol Today 1999; 15: 22–28.

26. Yarovinsky F., Zhang D., Andersen J.F., et al. TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 2005; 308: 1626–1629.

27. Denkers E.Y., Gazzinelli R.T.. Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection. Clin Microbiol Rev 1998; 11: 569–588.

28. Aliberti J.. Host persistence: exploitation of anti-inflammatory pathways by Toxoplasma gondii. Nature Rev Immunol 2005; 5: 162–170.

29. Petri W.A. Jr, Singh U.. Diagnosis and management of amebiasis. Clin Infect Dis 1999; 29: 1117–1125.

30. Stanley S.L. Jr. Amoebiasis. Lancet 2003; 361: 1025–1034.

31. Asgharpour A., Gilchrist C., Baba D., et al. Resistance to intestinal Entamoeba histolytica infection is conferred by innate immunity and Gr-1+ cells. Infect Immun 2005; 73: 4522–4529.

32. Huston C.D.. Parasite and host contributions to the pathogenesis of amebic colitis. Trends Parasitol 2004; 20: 23–26.

33. Li E., Stanley S.L. Jr. Protozoa. Amebiasis. Gastroenterol Clin North Am 1996; 25: 471–492.

34. Farthing M.J .. Giardiasis. Gastroenterol Clin North Am 1996; 25: 493–515.

35. Roxstrom-Lindquist K., Palm D., Reiner D., et al. Giardia immunity – an update. Trends Parasitol 2006; 22: 26–31.

36. Nash T.E., Merritt J.W. Jr, Conrad J.T.. Isolate and epitope variability in susceptibility of Giardia lamblia to intestinal proteases. Infect Immun 1991; 59: 1334–1340.

37. Li E., SL Stanley J.. Parasitic diseases: protozoa, 4th edn. Philadelphia: Lippincott: Williams & Wilkins, 2003.

38. Deng M., Rutherford M.S., Abrahamsen M.S.. Host intestinal epithelial response to Cryptosporidium parvum. Adv Drug Del Rev 2004; 56: 869–884.

39. Riggs M.W.. Recent advances in cryptosporidiosis: the immune response. Microbes Infect 2002; 4: 1067–1080.

40. Chai J.Y., Guk S.M., Han H.K., et al. Role of intraepithelial lymphocytes in mucosal immune responses of mice experimentally infected with Cryptosporidium parvum. J Parasitol 1999; 85: 234–239.

41. Draper D., Donohoe W., Mortimer L., et al. Cysteine proteases of Trichomonas vaginalis degrade secretory leukocyte protease inhibitor. J Infect Dis 1998; 178: 815–819.

42. Rendon-Maldonado J.G., Espinosa-Cantellano M., Gonzalez-Robles A., et al. Trichomonas vaginalis: in vitro phagocytosis of lactobacilli, vaginal epithelial cells, leukocytes, and erythrocytes. Exp Parasitol 1998; 89: 241–250.

43. Petrin D., Delgaty K., Bhatt R., et al. Clinical and microbiological aspects of Trichomonas vaginalis. Clin Microbiol Rev 1998; 11: 300–317.

44. Markell E ., John D ., Krotoski W .. Medical parasitology, 8th edn. Philadelphia: WB Saunders, 1999.

RE

FE

RE

NC

ES