A. Toll-like receptors (see Fig. 6.20), the mannose receptor (see Fig. 7.11) and scavenger receptors (see Fig. 7.10) allow phagocytes to directly recognize pathogens. Ficolins, collectins, and pentraxins act as soluble opsonins by binding to pathogen surfaces (see Fig. 6.w3).

A unifying feature of these targets is their highly conserved structures, which are invariant between parasites of a given class.

Although many parasites are known to activate the immune system in a non-specific manner shortly after infection, it is only recently that attention has been given to the mechanisms involved.

While major advances are being achieved in the area of microbial recognition by PRRs, a small but growing number of studies show that parasites also possess specific molecular patterns capable of engaging PRRs. Examples of some parasite PAMPs along with their receptors are given in Figure 15.6.

Fig. 15.6 Innate immune receptors involved in parasite recognition

Innate immune receptors involved in parasite recognition.

Toll-like receptors recognize parasite molecules

The discovery of the TLR family, an evolutionarily conserved group of mammalian PRRs involved in antimicrobial immunity, has enriched our understanding of how innate and adaptive immunity are mutually dependent.

A few studies have examined the role of TLRs in immunity to parasites. For example:

• T. gondii binds TLR11 via profilin and associates with TLR3, TLR7, and TLR9 via the endoplasmic reticulum protein UNC93B1;

• TLR9 mediates innate immune activation by the malaria pigment hemozoin;

• lyso-phosphatidylserine (lyso-PS) from S. mansoni, glycophosphatidylinositol (GPI) anchors, and Tc52 from T. cruzi are capable of signaling through TLR2.

Interestingly, TLR2 triggering by these diverse parasite patterns leads to different immune outcomes: for S. mansoni, triggering leads to the development of fully mature dendritic cells capable of inducing a Treg response (see Chapter 11), characterized by elevated IL-10 levels; for T. cruzi, mature dendritic cells induce a TH1 response (see Chapter 7) with raised levels of IL-12. This dichotomous response could, in part, be explained by the cooperation between TLR2 and other TLRs. However, there has been difficulty in assigning definitive contributions of TLRs to activation by specific parasite proteins since samples are easily contaminated, e.g. with bacterial PAMPs.

Classical human PRRs also contribute to recognition of parasites

Classical PRRs also play important roles in the innate response to parasite infection (see Fig. 15.6) and include collectins (e.g. MBL), pentraxins (e.g. CRP), C-type lectins (e.g. macrophage mannose receptor, and scavenger receptors (e.g. CD36) – see Chapters 6 and 7. For example, MBL binds mannose-rich LPG from Leishmania, Plasmodium, trypanosomes, and schistosomes; and polymorphisms in the MBL gene are associated with increased susceptibility to severe malaria.

Complement receptors are archetypal PRRs

Complement receptors, in particular CR3, are archetypal PRRs involved in innate immune responses (see Fig. 15.6). They are truly multifunctional, being involved in phagocyte adhesion, recognition, migration, activation, and microbe elimination.

Why then is CR3, a linchpin of phagocyte responses, the favored portal of entry for diverse intracellular parasites, including Leishmania via LPG?

• CR3 offers a multiplicity of binding sites, enabling opsonic or non-opsonic binding;

• phagocytosis by CR3 alone does not generate an oxidative burst in phagocytic cells;

• binding of CR3 suppresses the secretion of IL-12.

Q. What effect would suppression of IL-12 secretion have on the immune response?

A. Because IL-12 promotes the development of the TH1 response, reduced expression will tend to reduce macrophage-mediated immunity.

In isolation, CR3 is not an activating receptor. It requires cooperation from other receptors, most notably Fc receptors, for pathogen killing. Helminths have also exploited this chink in the immune armory. Hookworm NIF has been shown to bind a domain in the α subunit of CR3, presumably to downregulate cell-mediated immunity.

Adaptive immune responses to parasites

T and B cells are pivotal in the development of immunity

In most parasitic infections, protection can be conferred experimentally on normal animals by the transfer of spleen cells, especially T cells, from immune animals.

The T cell requirement is also demonstrable because nude (athymic) or T-deprived mice fail to clear otherwise non-lethal infections of protozoa such as T. cruzi or Plasmodium yoelii, and T cell-deprived rats fail to expel the intestinal worm Nippostrongylus brasiliensis (Fig. 15.7).

Fig. 15.7 Parasitic infections in T cell-deprived mice

The first two graphs plot the increase in number of blood-borne protozoa (parasitemia) following infection. (1) T. cruzi multiplies faster (and gives fatal parasitemia) in mice that have been thymectomized and irradiated to destroy T cells (Thym x). In normal mice, parasites are cleared from the blood by day 16. Reconstitution of T cell-deprived mice with T cells from immune mice restores their ability to control the parasitemia. In these experiments both thymectomized groups were given fetal liver cells to restore vital hematopoietic function. (2) P. yoelii causes a self-limiting infection in normal mice and the parasites are cleared from the blood by day 20. In nude mice the parasites continue to multiply, killing the mice after about 30 days. (3) This graph illustrates the time course of the elimination of the intestinal nematode N. brasiliensis from the gut of rats. In normal rats the worms are all expelled by day 13, as determined by the number of worm eggs present in the rats’ feces. T cells are necessary for this expulsion to occur, as shown by the establishment of a chronic infection in the gut of nude rats.

Counterintuitively, many parasites require signals from immune cells to thrive – for example, schistosomes fail to develop in the absence of hepatic CD4⁺ lymphocytes.

B cells also play key roles in regulating and controlling immunity to parasites. For example:

• B cells and antibodies are required for resistance to the parasitic gastrointestinal nematode Trichuris muris; and

• passive transfer of IgG can protect people from malaria.

Both CD4 and CD8 T cells are needed for protection from some parasites

The type of T cell responsible for controlling an infection varies with the parasite and the stage of infection, and depends upon the kinds of cytokine they produce. For example, CD4⁺ and CD8⁺ T cells protect against different phases of Plasmodium infection:

• CD4⁺ T cells mediate immunity against blood-stage P. yoelii;

• CD8⁺ T cells protect against the liver stage of Plasmodium berghei.

The action of CD8⁺ T cells is twofold:

• they secrete IFNγ, which inhibits the multiplication of parasites within hepatocytes;

• they are able to kill infected hepatocytes, but not infected erythrocytes.

Q. By what mechanism are the hepatocytes killed, and why do the CD8⁺ cells not recognize infected erythrocytes?

A. The CD8⁺ T cells recognize parasite antigens presented by MHC class I molecules and induce apoptosis in the target, via Fas ligand, tumor necrosis factor (TNFα), lymphotoxin, and granzymes. Erythrocytes do not express MHC molecules.

The immune response against T. cruzi depends not only upon CD4⁺ and CD8⁺ T cells, but also on NK cells and antibody production; the same is true for the immune response against T. gondii.

CD8⁺ T cells confer protection in mice depleted of CD4⁺ T cells, both through their production of IFNγ and because they are cytotoxic for infected macrophages.

NK cells, stimulated by IL-12 secreted by the macrophages, are another source of IFNγ – chronic infections are associated with reduced production of IFNγ.

These observations probably underlie the high incidence of toxoplasmosis in patients with AIDS, who are deficient in CD4⁺ T cells.

CD4⁺ T cells are critical for the expulsion of intestinal nematodes and as immunity to T. muris can be transferred to a SCID (severe combined immune deficiency) mouse by the transfer of CD4⁺ T cells alone there is no evidence for a role of CD8⁺ T cells.

The cytokines produced by CD4⁺ T cells can be important in determining the outcome of infection. As TH1 and TH2 cells have contrasting and cross-regulating cytokine profiles, the roles of TH1 or TH2 cells in determining the outcome of parasitic infections have been extensively investigated.

As a result of early studies, predominantly in mouse infections, certain dogmas have arisen suggesting that:

• TH1 responses mediate killing of intracellular pathogens; and

• TH2 responses eliminate extracellular ones.

However, this is very much an oversimplification of the true picture, and based on work in animal models that may not fully recapitulate the human immune system.

Although the TH1/TH2 paradigm may be a useful tool in some situations, it is probably more realistic in humans to consider that TH1 and TH2 phenotypes represent the extremes of a continuum of cytokine profiles, and that perhaps it may be more accurate to look at the role of the cytokines themselves in the resolution of infectious disease, particularly as new TH subsets are being discovered, e.g. TH17, TH22, and TH9, and their roles in immunity to parasites investigated.

Regulatory T cells are able to modulate the extremes of both TH2 and TH1 responses.

Cytokines, chemokines, and their receptors have important roles

Q. What experimental methods can be used to elucidate whether a particular cytokine is needed to clear a parasite infection?

A. Experiments with cytokine-knockout animals or cytokine blocking with specific antibody can determine whether a particular cytokine is required. This can be confirmed by observing whether exogenously administered cytokine speeds recovery.

Cytokines not only act on effector cells to enhance their cytotoxic or cytostatic capabilities, but also act as growth factors to increase cell numbers, while chemokines attract cells to the sites of infection. Thus in malaria, the characteristic enlargement of the spleen is caused by an enormous increase in cell numbers.

Other examples include:

• the accumulation of macrophages in the granulomas that develop in the liver in schistosomiasis;

• the eosinophilia characteristic of helminth infections; and

• the recruitment of eosinophils and mast cells into the gut mucosa that occurs in worm infections of the gastrointestinal tract.

Mucosal mast cells and eosinophils are both important in determining the outcome of some helminth infections and proliferate in response to the products of T cells – IL-3, granulocyte–macrophage colony stimulating factor (GM-CSF), and IL-5 respectively.

However, an increase in cell number can itself harm the host. Thus administration of IL-3 to mice infected with Leishmania major can exacerbate the local infection and increase the dissemination of the parasites, probably through the proliferation of bone marrow precursors of the cells the parasites inhabit.

IL-10 and transforming growth factor-β (TGFβ), the regulatory cytokines (see Chapter 11), downregulate the proinflammatory response and thus minimize pathological damage.

Chemokines are key molecules in recruiting immune cells by chemotaxis, but also act in leukocyte activation, hematopoiesis, inflammation, and antiparasite immunity.

Protozoan parasites have been most studied in the context of chemokines and their diverse roles in the parasite–host relationship. For example, T. gondii possesses cyclophilin-18, which binds to the chemokine receptor CCR5 and induces IL-12 production by dendritic cells.

T cell responses to protozoa depend on the species

T cell-mediated immunity operating to control protozoan parasites depends on the species of animal infected and the location and complexity of the parasite life cycle within the host.

For example, in mouse models, the induction of TH1 cells with concomitant upregulation of IFNγ and nitric oxide (NO^•) is crucial for protection of mice from Leishmania. Strains of mice driving TH2 responses on infection, manifested by high levels of IL-4, IL-13, IL-10, and antibody, develop progressive and ultimately lethal disease (Fig. 15.8).

Fig. 15.8 Development of the immune response to Leishmania major and Trichuris muris infection

The cytokines secreted by the different subsets of T cells and their effect on the resolution of the disease are shown. Note that resolution of infection is dependent on mouse strain. (Mø, macrophage.)

The polarization of TH cell responses in murine models does not conveniently translate to humans, where both TH1 and TH2 responses appear to be involved in protection.

The importance of TH1 cells for protection from toxoplasmosis is also evident in murine models.

For malaria, the TH1/TH2 paradigm is less helpful in understanding immunity, because the type of immune response mounted and the ensuing risk of pathology depends on whether the first exposure to the parasite occurs during infancy or adulthood. As a consequence, immunity to malaria is best thought of in the context of regulated TH1 responses. Thus, in endemic populations, primary malaria infections in infants induce low levels of IFNγ and TNFα via an innate pathway (potentially involving NK cells), which leads to T cell priming.

The infection induces minimal pathology and the parasites can be cleared, either immunologically via maternal antibody or because parasites fail to thrive in fetal hemoglobin. On reinfection, the malaria-primed T cells produce massive amounts of IFNγ and TNFα leading to an increased risk of unwanted pathology, including cerebral malaria.

Q. What effects will IFNγ and TNFα have on cerebral blood vessels?

A. These cytokines cause an increase in adhesion molecules (ICAM-1, VCAM-1) and synthesis of inflammatory chemokines (CCL2, CXCL10) producing leukocyte migration into the brain (see Chapter 6). They also cause an increase in the permeability of the vessels so that large serum molecules enter the CNS, and ionic equilibria are disturbed. This is referred to as a breakdown in the blood–brain barrier.

Further infections induce effective anti-parasite immunity principally through the development of an individual’s own repertoire of high-affinity antibodies, which inhibit parasite development. This change in immune environment ultimately leads to a switch in T cell phenotype from TH1 to a regulatory T cell phenotype in which raised levels of IL-10 and TGFβ can be detected.

By contrast, non-immune individuals who contract malaria for the first time in adulthood are unable to control their infections and are more likely to develop severe pathology. This is believed to arise from cross-reactively primed T cells generated against other microbes that appear to contribute to the development of severe disease.

The immune response to worms depends upon TH2-secreted cytokines

IgE and eosinophilia are the hallmarks of the immune response to worm infections, and depend upon cytokines secreted by TH2 cells (see Fig. 15.8).

In humans schistosomiasis and infection with gastrointestinal nematodes, resistance to reinfection after drug treatment is correlated with the production of IgE and high pre-treatment levels of TH2 cytokines such as IL-4, IL-5, and IL-13.

The primary stimuli for TH2 development in schistosomiasis are egg antigens. Similarly the excretory and secretory products of nematodes have been shown to polarize cells towards TH2 responses. Again the control of T cell phenotype seems to be exerted by the dendritic cell after exposure to these substances.

The mechanisms of induction of TH2 responses are less well understood than TH1 responses.

• one hypothesis, the default hypothesis, suggests that unless the triggers for TH1 responses are received (including high IL-12), TH2 responses occur;

• more recent evidence, however, suggests that specific signals induce the T cell to make TH2 cytokines, probably including cell–cell interactions.

The pattern of cytokine production in infected hosts may be different from that in vaccinated hosts. For example:

• in mice infected with S. mansoni, IL-5-producing TH2 cells predominate;

• in mice that have been immunized, IgE levels and eosinophil numbers are low and TH1 cells predominate.

IFNγ activates effector cells that destroy lung stage larvae, via the production of NO^•.

Q. How does IFNγ lead to the production of NO^•?

A. It causes the production of inducible NO^• synthase in macrophages (see Fig. 7.19).

However, when adult worms start to produce eggs, a soluble egg antigen is released that has an effect only in susceptible mice. The antigen reduces levels of IFNγ and increases production of IL-5.

TH2 cytokines control effector mechanisms important in controlling intestinal worm infections. Perhaps the example that demonstrates this most clearly is T. muris infection in mice.

• animals normally resistant to infection develop persistent infections in IL-4 and/or IL-13 knockout mice;

• conversely, susceptible mice expel the worms if IL-4 activity is promoted by administration of neutralizing antibody against IFNγ.

IL-9 is another TH2 cytokine that seems to be important in resistance to intestinal nematode infection and is involved in the production of mucosal mast cell responses and the production of IgE. IL-9 transgenic mice that produce higher levels of this cytokine have enhanced expulsion of T. muris.

What is clear from a number of studies is that there is no single mechanism by which a TH2 response mediates expulsion of all intestinal worms. The species of worm, its anatomical position within the gut, and the immune status of the host are all factors likely to influence whether a particular immune mechanism will be effective at promoting worm loss.

Some worm infections deviate the immune response

The role of IFNγ in promoting chronic infection is again shown by the administration of IL-12 to mice soon after infection with the intestinal worm Nippostrongylus braziliensis (Fig. 15.w2 ).

Fig. 15.w2 Course of infection with Nipppostrongylus braziliensis in mice

The normal course of infection in Balb/c mice is that worms will be expelled by day 11. This is associated with a TH2 response. If IL-12 is administered straight after infection a TH1 response is promoted and as a result more worms establish and the infection is maintained for longer. If IL-12 is administered, but IFNγ is neutralized, this result is reversed.

N. braziliensis stimulates IFNγ production, which delays expulsion of the worms.

IL-12 acts by inhibiting the production of TH2 cytokines – in particular IL-4 and IL-5, thereby preventing the production of IgE, eosinophilia, and mast cell hypertrophy.

The host may isolate the parasite with inflammatory cells

In some parasitic infections, the immune system cannot completely eliminate the parasite, but reacts by isolating the organism with inflammatory cells. The host reacts to locally released antigen, which stimulates the production of cytokines that recruit cells to the region. An example of this has been shown in mice vaccinated with radiation-attenuated schistosome cercariae. Infiltrating cells, which are mostly TH1-type lymphocytes, surround the lung-stage larvae as early as 24 hours after intravenous challenge infection. This prevents subsequent migration to the site necessary for development into the adult parasite.

The schistosome egg granuloma in the liver is another example of the host reacting by ‘walling off’ the parasite. This reaction is a chronic cell-mediated response to soluble antigens released by eggs that have become trapped in the liver. Macrophages accumulate and release fibrogenic factors, which stimulate the formation of granulomatous tissue and, ultimately, fibrosis. Although this reaction may benefit the host, in that it insulates the liver cells from toxins secreted by the worm eggs, it is also the major source of pathology, causing irreversible changes in the liver and the loss of liver function. In the absence of T cells, there is no granuloma formation and no subsequent fibrous encapsulation. Different mechanisms may affect:

• worms that inhabit different anatomical sites, such as the gut (e.g. T. trichura) or the tissues (e.g. Onchocerca volvulus); and

• different stages of the life cycle (e.g. schistosome larvae in the lungs and adult worms in the veins).

Parasites induce non-specific and specific antibody production

Many parasitic infections provoke a non-specific hyper-gammaglobulinemia, much of which is probably due to substances released from the parasites acting as B cell mitogens.

Levels of total immunoglobulins are raised:

• IgM in trypanosomiasis and malaria;

• IgG in malaria and visceral leishmaniasis.

The relative importance of antibody-dependent and antibody-independent responses varies with the infection and host (Fig. 15.9).

Fig. 15.9 Relative importance of antibody-dependent and antibody-independent responses in protozoal infections

This table summarizes the relative importance of the two immune responses, the mechanisms involved, and, for antibody, the means by which the protozoon can evade damage by antibody. Antibody is the most important part of the immune response against those parasites that live in the blood stream, such as African trypanosomes and malarial parasites, whereas cell-mediated immunity is active against those like Leishmania that live in the tissues. Antibody can damage parasites directly, enhance their clearance by phagocytosis, activate complement, or block their entry into their host cell and so limit the spread of infection. Once inside the cell the parasite is safe from the effects of antibody. Trypanosoma cruzi and Leishmania spp. are both susceptible to the action of oxygen metabolites released by the respiratory burst of macrophages, and to NO^•. Treating macrophages with cytokines enhances release of these products and diminishes the entry and survival of the parasites. (ADCC, antibody-dependent cell-mediated cytotoxicity.)

The mechanisms by which specific antibody can control parasitic infections and its effects are summarized in Figure 15.10. Antibody:

• can act directly on protozoa to damage them, either by itself or by activating the complement system (Fig. 15.11);

• can neutralize a parasite directly by blocking its attachment to a new host cell, as with Plasmodium spp., whose merozoites enter red blood cells through a special receptor – their entry is inhibited by specific antibody (Fig. 15.w3 );

• may prevent spread (e.g. in the acute phase of infection by T. cruzi);

• can enhance phagocytosis by macrophages – phagocytosis is increased even more by the addition of complement; these effects are mediated by Fc and C3 receptors on macrophages, which may increase in number as a result of macrophage activation;

• is involved in antibody-dependent cell-mediated cytotoxicity (ADCC), for example in infections caused by T. cruzi, T. spiralis, S. mansoni, and filarial worms – cytotoxic cells such as macrophages, neutrophils, and eosinophils adhere to antibody-coated worms by means of their Fc and C3 receptors and degranulate, spilling their toxic contents onto the worm (see Fig. 15.13).

Fig. 15.10 Mechanisms by which specific antibody controls some parasitic infections

(1) Direct damage. Antibody activates the classical complement pathway, causing damage to the parasite membrane and increasing susceptibility to other mediators. (2) Neutralization. Parasites such as Plasmodium spp. spread to new cells by specific receptor attachment; blocking the merozoite binding site with antibody prevents attachment to the receptors on the erythrocyte surface and prevents further multiplication. (3) Enhancement of phagocytosis. Complement C3b deposited on the parasite membrane opsonizes it for phagocytosis by cells with C3b receptors (e.g. macrophages). Macrophages also have Fc receptors. (4) Eosinophils, neutrophils, platelets, and macrophages may be cytotoxic for some parasites when they recognize the parasite via specific antibody (ADCC). The reaction is enhanced by complement.

Fig. 15.11 Direct effect of specific antibody on sporozoites of malaria parasites

These scanning electron micrographs show a sporozoite of P. berghei, which causes malaria in rodents, before (1) and after (2) incubation in immune serum. The surface of the sporozoite is damaged by the antibody, which perturbs the outer membrane, causing leakage of fluid. Specific antibody protects against infection with Plasmodium spp. at several of the extracellular stages of the life cycle. The antibody is stage specific in each case.

(Courtesy of Dr R Nussenzweig.)

Fig. 15.w3 Effect of antibody on malarial parasites

(1) Transfer of γ-globulin from immune adults to a child infected with Plasmodium falciparum caused a sharp drop in parasitemia. Specific antibody acts at the merozoite stage in the life of the parasite and prevents the initiation of further cycles of multiplication in the blood. The development of gametocytes from existing intracellular forms is unaffected. (2) In culture, the presence of immune serum blocks the continued increase in number of Plasmodium knowlesi (a malarial parasite of monkeys), as measured by incorporation of ³ H-leucine. It stops multiplication at the stage after schizont rupture by preventing the released merozoites from invading fresh red blood cells. The inhibitory activity of the immune serum can be reduced by prior absorption of the specific antibody with free schizonts.

Fig. 15.13 Eosinophil adhesion and degranulation

A schistosomule being killed by eosinophils from mouse bone marrow, cultured in the presence of IL-5. The larval helminth has been first treated with IgG and the eosinophils adhere by means of their Fcγ receptors.

(Courtesy of Dr C Sanderson.)

Different antibody isotypes may have different effects. In individuals infected with schistosomes, parasite-specific IgE and IgA are associated with resistance to infection and there is an inverse relationship between the amount of IgE in the blood and reinfection.

Q. What roles does IgE have in immune defense?

A. IgE mediates inflammation by binding to mast cells and basophils, sensitizing them to parasite antigens. Additionally it can act as an opsonin for eosinophils (see Chapter 3). Polyclonal activation of IgE production can inhibit cross-linking of specific IgE on mast cells by ‘crowding out’.

IgG4 appears to block the action of IgE; reinfection is more likely in children who have high levels of IgG4 and infection rates are highest in 10–14-year-olds when IgG4 levels are also at their highest. Class switching to IgG4 appears to occur in the context of a modified TH2 response involving the induction of Tregs.

In many infections it is difficult to distinguish between cell-mediated and antibody-mediated responses because both can act in concert against the parasite. This is illustrated in Figure 15.12, which summarizes the immune reaction that can be mounted against schistosome larvae.

Fig. 15.12 Possible effector responses to schistosomules

The various effector mechanisms damaging schistosomes in vitro are shown. Complement alone damages worms (1) and also does so in combination with antibody (2). TH1 cells may act directly, reducing the number of larvae in the lungs (3). Antibody sensitizes neutrophils (4), macrophages (5), platelets (6), and eosinophils (7) for ADCC. Neutrophils and macrophages probably act by releasing toxic oxygen and nitrogen metabolites, whereas eosinophils damage the worm tegument by the release of major basic protein (MBP) plus reactive oxygen intermediates (ROI). The response is potentiated by cytokines (e.g. TNFα). IgE antibody is important in sensitizing both eosinophils and local mast cells, which release a variety of mediators, including those that activate the eosinophils.

Immune effector cells

Macrophages, neutrophils, eosinophils, mast cells and platelets can all damage parasites. Antibody and cytokines produced specifically in response to parasite antigens enhance the antiparasitic activities of all these effector cells, though tissue macrophages, monocytes, and granulocytes have some intrinsic activity before enhancement. The point of entry of the parasite is obviously important, for example:

• the cercariae of S. mansoni enter through the skin – experimental depletion of macrophages, neutrophils, and eosinophils from the skin of mice increases their susceptibility to infection;

• trypanosomes and malarial parasites entering the blood are removed from the circulation by phagocytic cells in the skin, spleen and liver;

• comparison of strains of mice with various immunological defects for their resistance to infection by Trypanosoma rhodesiense shows that the African trypanosomes are destroyed by macrophages, and later in infection, when opsonized with antibodies and complement C3b, they are taken up by macrophages in the liver more quickly still.

Before acting as APCs initiating an immune response, macrophages act as effector cells to inhibit the multiplication of parasites or even to destroy them. They also secrete molecules that regulate the inflammatory response:

• some of these molecules – IL-1, IL-12, TNFα, and the colony stimulating factors (CSFs) – enhance immunity by activating other cells or stimulating their proliferation;

• others, like IL-10, prostaglandins, and TGFβ, may be anti-inflammatory and immunosuppressive.

Macrophages can kill extracellular parasites

Phagocytosis by macrophages provides an important defense against the smaller parasites. Macrophages also secrete many cytotoxic factors, enabling them to kill parasites without ingesting them.

When activated by cytokines, macrophages can kill both relatively small extracellular parasites, such as the erythrocytic stages of malaria, and also larger parasites, such as the larval stages of the schistosome. Macrophages also:

• act as killer cells through ADCC – specific IgG and IgE, for instance, enhance their ability to kill schistosomules;

• secrete cytokines, such as TNFα and IL-1, which interact with other types of cell, for example rendering hepatocytes resistant to malarial parasites.

Reactive oxygen intermediates (ROIs) are generated by macrophages and granulocytes following phagocytosis of T. cruzi, T. gondii, Leishmania spp., and malarial parasites, for instance. Filarial worms and schistosomes also stimulate the respiratory burst.

When activated by cytokines, macrophages release more superoxide and hydrogen peroxide than normal resident macrophages, and their oxygen-independent killing mechanisms are similarly enhanced.

Nitric oxide, a product of L-arginine metabolism, is another potent toxin. Its synthesis by macrophages in mouse experimental systems is induced by the cytokines IFNγ and TNFα and is greatly increased when they act synergistically. NO^• can also be produced by endothelial cells. It contributes to host resistance in leishmaniasis, schistosomiasis, and malaria, and is probably important in the control of most parasitic infections (see Fig. 15.13). For instance, the innate resistance to infection by T. gondii that is lost in immunocompromised individuals appears to be due to the inhibition of parasite multiplication by such an oxygen-independent mechanism.

Activation of macrophages is a feature of early infection

All macrophage effector functions are enhanced soon after infection. Although their specific activation is by cytokines secreted by T cells (e.g. IFNγ, GM-CSF, IL-3, and IL-4), they can also be activated by T cell-independent mechanisms, for example:

• NK cells secrete IFNγ when stimulated by IL-12 produced by macrophages;

• macrophages secrete TNFα in response to some parasite products (e.g. phospholipid-containing antigens of malarial parasites and some T. brucei antigens) – this TNFα then activates other macrophages.

Although TNFα may be secreted by several other cell types, activated macrophages are the most important source of TNFα, which is necessary for protective responses to several species of protozoa (e.g. Leishmania spp.) and helminths. Thus TNFα activates macrophages, eosinophils, and platelets to kill the larval form of S. mansoni, its effects being enhanced by IFNγ.

TNFα may be harmful as well as beneficial to the infected host, depending upon the amount produced and whether it is free in the circulation or locally confined. Serum concentrations of TNFα in falciparum malaria correlate with the severity of the disease. Administration of TNFα cures a susceptible strain of mice infected with the rodent malarial parasite Plasmodium chabaudi, but kills a genetically resistant strain. Presumably the latter can already make enough TNFα to control parasite replication, and any more has toxic effects.

Neutrophils can kill large and small parasites

The effector properties displayed by macrophages are also seen in neutrophils. Neutrophils are phagocytic and can kill by both oxygen-dependent and oxygen-independent mechanisms, including NO^•. They produce a more intense respiratory burst than macrophages and their secretory granules contain highly cytotoxic proteins.

Q. Which groups of cytotoxic protein are found in neutrophil granules?

A. Defensins, seprocidins, and cathelicidins (see Chapter 2).

Neutrophils can be activated by cytokines, such as IL-8, IFNγ, TNFα, and GM-CSF.

Extracellular destruction by neutrophils is mediated by hydrogen peroxide, whereas granular components are involved in the intracellular destruction of ingested organisms.

Neutrophils are present in parasite-infected inflammatory lesions and probably act to clear parasites from bursting cells.

Like macrophages, neutrophils bear Fc and complement receptors and can participate in antibody-dependent cytotoxic reactions to kill the larvae of S. mansoni, for example. In this mode, they can be more destructive than eosinophils against several species of nematode, including T. spiralis, though the relative effectiveness of the two types of cell may depend upon the isotype and specificity of antibody present.

Eosinophils are characteristically associated with worm infections

It has been suggested that:

• the eosinophil evolved specifically as a defense against the tissue stages of parasites that are too large to be phagocytosed;

• the IgE-dependent mast cell reaction has evolved primarily to localize eosinophils near the parasite and enhance their antiparasitic functions.

The importance of eosinophils in vivo has been shown by experiments using antiserum against eosinophils. Mice infected with T. spiralis and treated with the antiserum develop more cysts in their muscles than the controls – without the protection offered by eosinophils, the mice cannot eliminate the worms and so encyst the parasites to minimize damage.

However, recent work has shown that although eosinophils can help the host to control a worm infection, particularly by limiting migration through the tissues, they do not always do so. For instance, their removal does not abolish the immunity of mice infected with S. mansoni, nor does this increase the parasite load in a tapeworm infection.

Removal of IL-5, which is important in the generation and activation of eosinophils, did not change the outcome of T. spiralis or T. muris infection. In contrast the infectivity of Strongyloides venezuelensis is enhanced in IL-5-deficient mice. Although T. spiralis worm burdens were not affected in a primary infection of IL-5 deleted mice, the worm numbers were significantly higher after challenge infection.

The role of IL-5 and therefore eosinophils has also been suggested from human epidemiological studies on gastrointestinal nematode infections where, after drug treatment, low reinfection worm burdens were associated with high pre-treatment levels of IL-5.

Elevated eosinophilia is often associated with high levels of IgE, both of which are hallmarks of infection with parasites. Although eosinophils express FcεRI, most of the protein is confined to the cytoplasm, and there is little evidence for IgE-dependent function.

Eosinophils can kill helminths by oxygen-dependent and independent mechanisms

Eosinophils are less phagocytic than neutrophils. They degranulate in response to perturbation of their surface membrane and their activities are enhanced by cytokines such as TNFα and GM-CSF. Most of their activities, however, are controlled by antigen-specific mechanisms. Thus their binding in vitro to the larvae of worms (e.g. S. mansoni and T. spiralis) increases the release of their granular contents onto the surface of the worms (Fig. 15.13).

Damage to schistosomules can be caused by the major basic protein (MBP) of the eosinophil crystalloid core. MBP is not specific for any particular target, but because it is confined to a small space between the eosinophil and the schistosome, there is little damage to nearby host cells.

Eosinophils and mast cells can act together

The killing of S. mansoni larvae by eosinophils is enhanced by mast cell products, and when studied in vitro eosinophils from patients with schistosomiasis are found to be more effective than those from normal subjects. The antigens released cause local IgE-dependent degranulation of mast cells and the release of mediators. These selectively attract eosinophils to the site and further enhance their activity. Other products of eosinophils later block the mast cell reactions. These effector mechanisms may function in vivo, as has been shown in monkeys, where schistosome killing is associated with eosinophil accumulation.

Mast cells control gastrointestinal helminths

In the case of T. spiralis and Heligmosomoides polygyrus there is good evidence to suggest the involvement of mucosal mast cells (Fig. 15.14)

Fig. 15.14 Section through the gut of a mouse infected with Heligmosomoides polygyrus

(1) Gut of an uninfected mouse. (2) Gut of an infected mouse. The crypts have shortened and a large influx of mast cells can be clearly seen.

Following mast cell activation, the mast cell contents are released resulting in changes to the permeability of the intestinal epithelium and ultimately an environment that appears hostile for continued T. spiralis survival. By contrast, expulsion of N. braziliensis and T. muris still proceeds normally following depression of mastocytosis, suggesting that the mast cell is not the major effector cell type.

Therefore, although TH2 cytokines are critical for the elimination of worms from the gut, the exact effector mechanism may vary.

Platelets can kill many types of parasite

Potential targets for platelets include the larval stage of flukes, T. gondii, and T. cruzi.

Like other effector cells, the cytotoxic activity of platelets is enhanced by treatment with cytokines (e.g. IFNγ and TNFα). In rats infected with S. mansoni, platelets become larvicidal when acute phase reactants appear in the serum but before antibody can be detected. Incubation of normal platelets in such serum can cause their activation.

Platelets, like macrophages and the other effector cells, also bear Fcε and Fcγ receptors, by which they mediate antibody-dependent cytotoxicity associated with IgE.

Parasite escape mechanisms

It is a necessary characteristic of all successful parasitic infections that they can evade the full effects of their host’s immune responses. Parasites have developed many different ways of doing this. Some even exploit cells and molecules of the immune system to their own advantage – Leishmania parasites, by using complement receptors to gain entry into macrophages, avoid triggering the oxidative burst and thus destruction by its toxic products.

Despite their protective role in the immune response to many different parasites:

• host TNFα actually stimulates egg production by adult worms of S. mansoni;

• IFN is used as a growth factor by T. brucei.

Parasites can resist destruction by complement

In the case of Leishmania, resistance correlates with virulence:

• L. tropica, which is easily killed by complement, causes a localized self-healing infection in the skin; whereas

• L. donovani, which is ten times more resistant to complement, becomes disseminated throughout the viscera, causing a disease that is often fatal.

The mechanisms whereby parasites can resist the effect of complement differ:

• the lipophosphoglycan (LPG) surface coat of L. major activates complement, but the complex is then shed so the parasite avoids lysis;

• the trypomastigotes of T. cruzi bear a surface glycoprotein that has activity resembling the decay accelerating factor (DAF) that limits the complement reaction. The resistance that schistosomules acquire as they mature is also correlated with the appearance of a surface molecule similar to DAF.

Intracellular parasites can avoid being killed by oxygen metabolites and lysosomal enzymes

Intracellular parasites that live inside macrophages have evolved different ways of avoiding being killed by oxygen metabolites and lysosomal enzymes (Fig. 15.15):

• T. gondii penetrates the macrophage by a non-phagocytic pathway and so avoids triggering the oxidative burst;

• Leishmania spp. can enter by binding to complement receptors – another way of avoiding the respiratory burst.

Fig. 15.15 The different ways by which protozoa that multiply within macrophages escape digestion by lysosomal enzymes

T. gondii – live parasites enter the cell actively into a membrane-bound vacuole. They are not attacked by enzymes because lysosomes do not fuse with this vacuole. Dead parasites, however, are taken up by normal phagocytosis into a phagosome (by interaction with the Fc receptors on the macrophage if they are coated with antibody) and are then destroyed by the enzymes of the lysosomes that fuse with it. T. cruzi – survival of these parasites depends upon their stage of development; trypomastigotes escape from the phagosome and divide in the cytoplasm whereas epimastigotes do not escape and are killed. The proportion of parasites found in the cytoplasm is decreased if the macrophages are activated. Leishmania spp. – These parasites multiply within the phagosome and the presence of a surface protease helps them resist digestion. If the macrophages are first activated by cytokines, the number of parasites entering the cell and the number that replicate diminish.

Leishmania organisms also possess enzymes such as superoxide dismutase, which protects them against the action of oxygen radicals.

It can be demonstrated that the vacuole in which Leishmania organisms survive is lyosomal in nature (Fig. 15.16), but the parasites have evolved mechanisms that protect it against enzymatic attack. The LPG surface coat acts as a scavenger of oxygen metabolites and affords protection against enzymatic attack, but a glycoprotein, Gp63 (Fig 15.w4 ) inhibits the action of the macrophage’s lysosomal enzymes.

Fig. 15.16 The leishmanial vacuole is lysosomal in nature

(1) Immunofluorescence of Leishmania mexicana-infected murine macrophages probed with a rhodamine-conjugated anti-tubulin antibody to illustrate the parasite (stained yellow/red) and a fluorescein-conjugated monoclonal antibody, which reacts with the late endosomal/lysosomal marker LAMP-1 (stained green). (2) Immunoelectron micrograph of L. mexicana-infected murine macrophage probed with gold-labeled anti-cathepsin D demonstrating the lysosomal aspartic proteinase in the leishmanial vacuole.

(Courtesy of Dr David Russell.)

Fig. 15.w4 Two surface antigens of Leishmania

Schematic representation of two surface antigens of Leishmania that are anchored to the membrane by phosphatidylinositol tails (GPI anchors). (1) This protein antigen, Gp63, has protease activity. That of L. mexicana, together with lipophosphoglycan (LPG), binds complement. This enables the promastigote to enter the macrophage through the C3 complement receptor. (2) This glycolipid antigen, a LPG, imparts resistance to complement-mediated lysis. That of L. major binds C3b, the third component of complement, enabling the promastigote to enter through the CR1 complement receptor. Antibodies to both antigens confer protection against murine cutaneous leishmaniasis. Note that many coat proteins of parasites, such as the variable surface glycoprotein (VSG) of T. brucei, are now known to be bound to the surface membrane by a GPI anchor.

Leishmania spp. can also downregulate the expression of MHC class II molecules on the macrophages they inhabit, thus reducing their capacity to stimulate TH cells.

These escape mechanisms, however, are less efficient in the immune host.

Q. Why would reduction in MHC molecule expression be less effective in an immune host?

A. In immune hosts, the release of IFNγ enhances MHC molecule expression by APCs. In addition the level of stimulation required by a primed T cell is less than for a naive T cell, due in part to its enhanced level of receptors for co-stimulatory signals (see Chapter 8).

Parasites can disguise themselves

Parasites that are vulnerable to specific antibody have evolved different methods of evading its effects.

African trypanosomes and malaria undergo antigenic variation

The molecule that forms the surface coat of the African trypanosome, the variable surface glycoprotein (VSG), changes to protect the underlying surface membrane from the host’s defense mechanisms. New populations of parasites are antigenically distinct from previous ones (Fig. 15.17).

Fig. 15.17 Antigenic variation in trypanosomes

Trypanosome infections may run for several months giving rise to successive waves of parasitemia. The graph shows a chart of the fluctuation in parasitemia in a patient with sleeping sickness. Although infection was initiated by a single parasite, each wave is caused by an immunologically distinct population of parasites (a, b, c, d); protection is not afforded by antibody against any of the preceding variants. There is a strong tendency for new variants to appear in the same order in different hosts. The micrographs show immunofluorescent labeling of trypanosomes with a variant antigen-type specific monoclonal antibody (left). The panel on the right shows the same field but with the nuclei and kinetoplasts of all the parasites stained with a dye that binds to DNA. Only some of the parasites express a given antigen variant.

(Courtesy of Dr Mike Turner.)

Several antigens of malarial parasites also undergo antigenic variation.

For example, the P. falciparum erythrocyte membrane protein-1 (PfEMP1) is extremely polymorphic and variable between different strains of the parasite because it is perpetually exposed to the immune system by its location on the red cell membrane. PfEMP1 can bind numerous host immune proteins, but particularly scavenger receptors, e.g. CD36 and scavenging antibodies that eliminate apoptotic, or damaged cells, e.g. natural IgM (see Fig. 15.20).

Fig. 15.20 Malarial pathology resulting from interactions with PfEMP1

Malaria-infected erythrocytes cause disease through many mechanisms involving the interaction between P. falciparum erythrocyte membrane protein-1 (PfEMP1) and diverse host receptors.

Other parasites acquire a surface layer of host antigens

Schistosomes acquire a surface layer of host antigens so that the host does not distinguish them from ‘self’. Schistosomules cultured in medium containing human serum and red blood cells can acquire surface molecules containing A, B, and H blood group determinants. They can also acquire MHC molecules and immunoglobulins. However, schistosomules maintained in a medium devoid of host molecules also become resistant to attack by antibody and complement, as mentioned above.

Some extracellular parasites hide from or resist immune attack

Some species of protozoa (e.g. Entamoeba histolytica) and helminths (e.g. T. spiralis) form protective cysts, while adult worms of Onchocerca volvulus in the skin induce the host to surround them with collagenous nodules.

Intestinal nematodes and tapeworms are preserved from many host responses simply because they live in the gut.

There are numerous examples of simple, physical, protective strategies in parasites:

• nematodes have a thick extracellular cuticle, which protects them from the toxic effects of an immune response;

• the tegument of schistosomes thickens during maturation to offer similar protection;

• the loose surface coat of many nematodes may slough off under immune attack;

• tapeworms actually prevent attack by secreting an elastase inhibitor, which stops them attracting neutrophils.

Many parasitic worms have evolved methods of resisting the oxidative burst. For instance, schistosomes have surface-associated glutathione S-transferases, and Onchocerca spp. can secrete superoxide dismutase.

Some nematodes and trematodes have evolved an elegant method of disabling antibodies by secreting proteases, which cleave immunoglobulins, removing the Fc portion, and preventing their interaction with Fc receptors on phagocytic cells; for example, schistosomes can cleave IgE.

Most parasites interfere with immune responses for their benefit

Parasites produce molecules that interfere with host immune function

Parasites produce molecules that can affect the phenotype of the adaptive response, which may be to their own advantage (Figs 15.18 and 15.19).

Fig. 15.18 Some mechanisms by which parasites avoid host immunity

A summary of the various methods that parasites have evolved to avoid host defense mechanisms.

Fig. 15.19 Some immunomodulatory effects of parasites

Interference with the host’s immune response by molecules released by protozoa or worms. Parasite products act via the APC to (1) interfere with antigen processing or presentation (e.g. protease inhibitors from filarial parasites interfere with proteases in the MHC signaling pathway and block antigen presentation); (2) induce suppressor macrophages, which can inhibit T cell proliferation; (3) induce regulatory T cells (e.g. lyso-PS from schistosomes acts via TLR2 on dendritic cells to induce T cells that secrete IL-10, which inhibits the inflammatory response). Parasite products may also affect lymphocytes to (3) make them become regulatory; (4) induce T or B cell tolerance by clonal exhaustion or by the induction of anergy; (5) cause polyclonal activation (many parasite products are mitogenic to T or B cells, and the high serum concentrations of non-specific IgM [and IgG] commonly found in parasitic infections probably result from this polyclonal stimulation – its continuation is believed to lead to impairment of B cell function, the progressive depletion of antigen-reactive B cells, and thus immunosuppression); (6) directly inhibit the activation of T or B cells (e.g. ES-62, a secreted product of a filarial parasite, is able to inhibit the proliferation of both T and B cells).

In leishmaniasis, T cells from patients infected with L. donovani when cultured with specific antigen do not secrete IL-2 or IFNγ. Their production of IL-1 and expression of MHC class II molecules is also decreased, whereas secretion of prostaglandins is increased. IL-2, characteristic of TH1 responses, is also deficient in other protozoal infections including malaria, African trypanosomiasis, and Chagas’ disease. In mice infected with T. cruzi, a parasite product appears to interfere with expression of the IL-2 receptor.

Filarial worms secrete a protease inhibitor that has been shown to affect the proteases critical in the processing of antigens to peptides resulting in the reduction of class II molecule presentation in filariasis. Onchocystatin – one such protease inhibitor – is also able to modulate T cell proliferation and elicit the upregulation of IL-10 expression and is therefore able to modulate the T cell phenotype. Prostaglandins (PGs) produced by helminth parasites may also perform a similar role by modulating APC function. PGE₂ is produced by filarial parasites and tapeworms and blocks the production of IL-12 by dendritic cells and thus may direct responses towards TH2.

Phosphorylcholine (PC)-containing molecules are commonly found in infectious organisms and experiments using a nematode PC-bearing glycoconjugate, ES-62, have been shown to desensitize APCs to subsequent exposure to LPS and may therefore also skew against a TH1 response (LPS is a classical inducer of TH1 responses). ES-62 is able to inhibit proliferation of T cells and B cells and inhibit IgE-mediated mast cell responses.

Parasites also produce cytokine-like molecules mimicking TGFβ, migration inhibitory factor (MIF), and a histamine-releasing factor.

Genes encoding possible cytokine homologs are being found as part of the genome sequencing projects that are under way for many parasites. Although the sequences are related to cytokines or cytokine receptors, their functions remain to be established.

Soluble parasite antigens released in huge quantities may impair the host’s response by a process termed immune distraction. Thus the soluble antigens (S or heat-stable antigens) of P. falciparum are thought to mop up circulating antibody, providing a ‘smokescreen’ and diverting the antibody from the body of the parasite.

Many of the surface antigens that are shed are soluble forms of molecules inserted into the parasite membrane by a GPI anchor, including the VSG of T. brucei, the LPG or ‘excreted factor’ of Leishmania (see Fig. 15.w4) and several surface antigens of schistosomules. These are released by endogenous phosphatidylinositol-specific phospholipases.

The hypergammaglobulinemic immunoglobulins induced by malaria parasites can bind to FcγRIIB, which may benefit the parasite.

Q. Which cells express FcγRIIB and what effect does ligation of this receptor have on them?

A. B cells express the receptor and ligation inhibits B cell function.

Some parasites suppress inflammation or immune responses

Immunosuppression is a common feature of chronic helminth infections, both parasite-specific and generalized. For example, patients with schistosomiasis and filariasis have diminished responsiveness to antigens from the infecting parasite. Studies have also shown diminished responses to bystander infections and vaccinations. This spillover suppression may in fact be beneficial to the host in some situations. For example, reduced inflammatory responses have been observed in Helicobacter pylori infection with malaria.

Parasites have co-evolved with humans over millions of years and until recently it was normal for people to carry worms, a fact that argues for their importance in the ‘hygiene hypothesis’, which proposes that the rise in immune disorders, including allergies and autoimmune illness, is due to cleaner living conditions and the almost complete elimination of parasitic infections in westernized societies.

The ability of parasites to suppress hyperactive immune responses is believed to be due to the induction of regulatory T cells (see Fig. 15.19). Understanding how regulatory T cells, also known as suppressor T cells, are induced and how they dampen immune responses is the focus of intensive research. It seems to be the dendritic cell that polarizes the T cell towards a regulator phenotype after exposure to parasite extracts.