Chapter 78Protozoa: Pathogenesis and Defenses

Seed JR.

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General Concepts

Resistance

Resistance is the ability of a host to defend itself against a pathogen. Resistance to protozoan parasites involves three interrelated mechanisms: nonspecific factors, cellular immunity, and humoral immunity.

Pathology

Protozoal infection results in tissue damage leading to disease. In chronic infections the tissue damage is often due to an immune response to the parasite and/or to host antigens as well as to changes in cytokine profiles. Alternatively, it may be due to toxic protozoal products and/or to mechanical damage.

Escape Mechanisms

Escape mechanisms are strategies by which parasites avoid the killing effect of the immune system in an immunocompetent host. Escape mechanisms used by protozoal parasites include the following.

Antigenic Masking: Antigenic masking is the ability of a parasite to escape immune detection by covering itself with host antigens.

Blocking of Serum Factors: Some parasites acquire a coating of antigen-antibody complexes or noncytotoxic antibodies that sterically blocks the binding of specific antibody or lymphocytes to the parasite surface antigens.

Intracellular Location: The intracellular habitat of some protozoan parasites protects them from the direct effects of the host's immune response. By concealing the parasite antigens, this strategy also delays detection by the immune system.

Antigenic Variation: Some protozoan parasites change their surface antigens during the course of an infection. Parasites carrying the new antigens escape the immune response to the original antigens.

Immunosuppression: Parasitic protozoan infections generally produce some degree of host immunosuppression. This reduced immune response may delay detection of antigenic variants. It may also reduce the ability of the immune system to inhibit the growth of and/or to kill the parasites.

Introduction

Resistance to parasitic protozoa appears to be similar to resistance against other infectious agents, although the mechanisms of resistance in protozoan infections are not yet as well understood. Resistance can be divided into two main groups of mechanisms: (1) nonspecific mechanism(s) or factor(s) such as the presence of a nonspecific serum component that is lethal to the parasite; and (2) specific mechanism(s) involving the immune system (Fig. 78-1). Probably the best studied nonspecific mechanisms involved in parasite resistance are the ones that control the susceptibility of red blood cells to invasion or growth of plasmodia, the agents of malaria. Individuals who are heterozygous or homozygous for the sickle cell hemoglobin trait are considerably more resistant to Plasmodium falciparum than are individuals with normal hemoglobin. Similarly, individuals who lack the Duffy factor on their red blood cells are not susceptible to P vivax. Possibly both the sickle cell trait and absence of the Duffy factor have become established in malaria-endemic populations as a result of selective pressure exerted by malaria. Epidemiologic evidence suggests that other inherited red blood cell abnormalities, such as thalassanemia and glucose-6-phosphate dehydrogenase deficiency, may contribute to survival of individuals in various malaria-endemic geographical regions. A second well-documented example of a nonspecific factor involved in resistance is the presence in the serum of humans of a trypanolytic factor that confers resistance against Trypanosoma brucei brucei, an agent of trypanosomiasis (sleeping sickness) in animals. There is evidence that other nonspecific factors, such as fever and the sex of the host, may also contribute to the host's resistance to various protozoan parasites. Although nonspecific factors can play a key role in resistance, usually they work in conjunction with the host's immune system (Fig. 78-1).

Figure 78-1. Some interrelationships between host factors involved in resistance to protozoan infections.

Figure 78-1

Some interrelationships between host factors involved in resistance to protozoan infections.

Different parasites elicit different humoral and/or cellular immune responses. In malaria and trypanosome infections, antibody appears to play a major role in immunity. In both T cruzi and T brucei gambiense infections, antibody-dependent cytotoxic reactions against the parasite have been reported. Although antibody has been shown to be responsible for clearing the African trypanosomes from the blood of infected animals, recent evidence suggests that the survival time of infected mice does not necessarily correlate with the ability of the animal to produce trypanosome-specific antibody. In other words, resistance as measured by survival time may not solely involve the specific humoral immune system. Recent data suggest that cellular immunity is required for resistance to malaria. for example, vaccine trials with a sporozoite antigen indicated that both an active cellular response and sporozoite-specific antibody may be needed for successful immunization.

Cellular immunity is believed to be the single most important defense mechanism in leishmaniasis and toxoplasmosis. In animals infected with Toxoplasma, the activated macrophage has been shown to play an important role in resistance. Accordingly, resistance to the protozoan parasites most likely involves nonspecific factors as well as specific humoral and/or cellular mechanisms. Cytokines are involved in the control of both the immune response and pathology. It has become apparent that there are subsets of both helper (h) and cytotoxic (c) T-cells that produce different profiles of cytokines. For example, the Th-1 subset produces gamma interferon (IFN-α), and interleukin-2 (IL-2) and is involved in cell-mediated immunity. In contrast the Th-2 subset produces IL-4 and IL-6, and is responsible for antibody-mediated immunity. The induction of a particular T-cell subset is key to recovery and resistance. The Th-1 subset and increased IFN-g are important in resistance to Leishmania, T cruzi and Toxoplasma infections, whereas the Th-2 response is more important in parasitic infections in which antibody is a key factor. It is important to recognize that the cytokines produced by one T-cell subset can up or downregulate the response of other T-cell subsets. IL-4 will downregulate Th-1 cells and exacerbate infection and/or susceptibility of mice to Leishmania. The cytokines produced by T and other cell types do not act directly on the parasites but influence other host cell types. The response of cells to cytokines includes a variety of physiological changes, such as changes in glucose, fatty acid and protein metabolism. For example, IL-1 and tumor necrosis factor will increase gluconeogenesis, and glucose oxidation. It should be noted that cytokines influence the metabolism not only of T-cells, but also a variety of other cell types and organ systems. Cytokines can also stimulate cell division and, therefore, clonal expansion of T and B-cell subsets. This can lead to increased antibody production and/or cytotoxic T-cell numbers. The list of cytokines and their functions is growing rapidly, and it would appear that these chemical messages influence all phases of the immune response. they are also clearly involved in the multitude of physiological responses (fever, decreased food intake, etc.) observed in an animal's response to a pathogen, and in the pathology that results.

Unlike most viral and bacterial infections, protozoan diseases are often chronic, lasting months or years. When associated with a strong host immune response, this type of chronic infection is apt to result in a high incidence of immunopathology. The question also arises of how these parasites survive in an immunocompetent animal. The remainder of this chapter treats the mechanisms responsible for pathology, particularly immunopathology, in protozoan disease, and the mechanisms by which parasites evade the immune responses of the host. Finally, because of the very rapid advances in our knowledge of the host-parasite relationship (due primarily to the development of techniques in molecular biology), it is necessary to briefly mention the potential for developing vaccines to the pathogenic protozoa.

Pathology

The protozoa can elicit humoral responses in which antigen-antibody complexes in the region of antibody excess activate Hageman blood coagulation factor (Factor XII), which in turn activates the coagulation, fibrinolytic, kinin and complement systems. It has been suggested that this type of immediate hypersensitivity is responsible for various clinical syndromes in African trypanosomiasis, including blood hyperviscosity, edema, and hypotension. Similar disease mechanisms would be expected in other infections by protozoa involving a strong humoral immune response (Table 78-1).

Table 78-1. Some Pathologic Mechanisms in Protozoal Diseases.

Table 78-1

Some Pathologic Mechanisms in Protozoal Diseases.

Immune complexes have been found circulating in serum and deposited in the kidneys and other tissues of humans and animals infected with protozoans. These parasite antigen-antibody complexes, plus complement, have been eluted from kidney tissue in cases of malaria and African trypanosomiasis. Antigen and antibody have been directly visualized in the glomeruli of infected animals by light and electron microscopy. Inflammatory cell infiltrates accompany these deposits, and signs of glomerulonephritis are usually seen. African trypanosomes and presumably their antigens are also found in a variety of extravascular locations. Immune complexes, cellular infiltrates, and tissue damage have been detected in these tissues.

Another important form of antibody-mediated pathology is autoimmunity. Autoantibodies to a number of different host antigens (for example, red blood cells, laminin, collagen, and DNA) have been demonstrated. These autoantibodies may play a role in the pathology of parasitic diseases in two ways. First the antibodies may exert a direct cytotoxic effect on the host cells; for example, autoantibodies that coat red blood cells produce hemolytic anemia. Alternatively, autoantibodies may be pathogenic through a buildup of antigen-antibody complexes in the kidneys or other tissues, leading to glomerulonephritis or other forms of immediate hypersensitivity. A particularly good example of a protozoan infection in which autoimmunity appears to be an important contributor to pathogenesis is T cruzi infection. In this case, there is substantial evidence that host and parasite share cross-reacting antigens. Antibodies and cytotoxic lymphocytes to these antigens appear to be harmful to host tissue. This type of experimental data, combined with the fact that the parasite itself seems not to cause the tissue pathology, lead one to conclude that autoimmunity may play a key role in pathogenesis.

Cellular hypersensitivity is also observed in protozoan diseases (Table 78-1). For example, in leishmaniasis (caused by Leishmania tropica), the lesions appear to be caused by a cell-mediated immune response and have many, if not all, of the characteristics of granulomas observed in tuberculosis or schistosomiasis. In these lesions, a continuing immune response to pathogens that are able to escape the host's defense mechanisms causes further influx of inflammatory cells, which leads to sustained reactions and continued pathology at the sites of antigen deposition. During a parasitic infection, various host cell products (cytokines, lymphokines, etc.) are released from activated cells of the immune system. These mediators influence the action of other cells and may be directly involved in pathogenesis. An example is tumor necrosis factor (TNF), which is released by lymphocytes. TNF may be involved in the muscle wasting observed in the chronic stages of African trypanosomiasis. TNF has also been implicated in the cachexia and wasting in Leishmania donovani infection, cerebral malaria in P falciparum in children and decreased survival in T cruzi-infected mice. It is apparent that mediators involved in resistance to protozoan parasites may also lead to pathology during a chronic infection (Fig. 78-1). There appears to be a delicate balance between the factors involved in resistance to infectious agents and those which ultimately produce pathology and clinical disease.

Numerous authors have suggested that toxic products produced by parasitic protozoa are responsible for at least some aspects of pathology (Table 78-1). For example, the glycoproteins on the surface of trypanosomes have been found to fix complement. This activation of complement presumably results in the production of biologically active and toxic complement fragments. In addition, trypanosomes are known to release proteases and phospholipases when they lyse. These enzymes can produce host cell destruction, inflammatory responses, and gross tissue pathology. Furthermore, it has been hypothesized that the trypanosomes contain a B-cell mitogen that may alter the immune response of the host by eliciting a polyclonal B-cell response that leads to immunosuppression. Finally it has recently been shown that the African trypanosomes also contain an endotoxin which is presumably released during antibody- mediated lysis. Parasitic protozoa have also been reported to synthesize (or contain) low-molecular-weight toxins. For example, the trypanosomes produce several indole catabolites; at pharmacologic doses, some of these catabolites can produce pathologic effects, such as fever, lethargy, and even immunosuppression. Similarly, enzymes, B-cell mitogen, etc., are presumably released by many if not all of the other parasitic protozoa. There has been limited work on the role of these protozoal products in pathogenesis. However, parasitic protozoa are generally not known to produce toxins with potencies comparable to those of the classic bacterial toxins (such as the toxins responsible for anthrax and botulism). One possible exception is the African trypanosomes which are suggested to contain an endotoxin.

Immune Escape

Parasite escape mechanisms may include a number of different phenomena (Table 78-2). In antigenic masking, the parasite becomes coated with host components and so fails to be recognized as foreign. In blocking, noncytotoxic antibody combines with parasite antigens and inhibits the binding of cytotoxic antibodies or cells. The parasite may pass part of its life cycle in an intracellular location, for example, in erythrocytes or macrophages, in which it is sheltered from intracellular digestion and from the cytotoxic action of antibody and/ or lymphocytes. Some parasites practice antigenic variation, altering their surface antigens during the course of an infection and thus evading the host's immune responses. Finally, the parasite may cause immunosuppression, reducing the host's immune response either to the parasite specifically or to foreign antigens in general. These strategies are discussed in more detail below.

Table 78-2. Some Proposed Immune Escape Mechanisms Used by Protozoan Parasites.

Table 78-2

Some Proposed Immune Escape Mechanisms Used by Protozoan Parasites.

Masking and Mimicry

Various species of trypanosomes have host immunoglobulins associated with their cell surfaces. There are several reports that these antibodies are not bound to the trypanosomes through their variable regions, but presumably through the Fc portion of their molecule. These antibodies may mask the parasite-that is, prevent immune recognition by the host. However, no evidence other than the presence of immunoglobulins on the surface of the trypanosomes supports this hypothesis. Mimicry, in which the parasite has the genetic information to synthesize antigens identical to those of its host, has not been demonstrated in parasitic protozoa.

Blocking

It has been hypothesized that in some cases antigen-antibody complexes in serum of infected animals bind to the parasite's surface, mechanically blocking the actions of cytotoxic antibodies or lymphocytes and directly inhibiting the actions of lymphocytes. This type of immune escape mechanism has been proposed for tumor cells and for the parasitic helminths. Because the trypanosomes carry immunoglobulins on their cell surfaces, they may use a similar mechanism; however, no direct evidence has yet been reported.

Intracellular location

Many protozoan parasites grow and divide within host cells. For example, Plasmodium parasites grow first in hepatocytes and then in red blood cells. Leishmania and Toxoplasma organisms are capable of growing in macrophages; one genus of parasitic protozoa, Theilera, not only multiplies in lymphocytes but appears even to stimulate the multiplication of the infected lymphocytes. Although some parasites, such as Plasmodium, are restricted to a limited number of host cell types, others, such as T cruzi and Toxoplasma, appear to be able to grow and divide in a variety of different host cells.

An intracellular refuge may protect a parasite from the harmful or lethal effects of antibody or cellular defense mechanisms. For example, Plasmodium may be susceptible to the actions of antibody only during the brief extracellular phases of its life cycle (the sporozoite and merozoite stages). It should be remembered that Plasmodium actually resides in a membrane-bound vacuole in the host cell. Thus, plasmodia are shielded from the external environment by at least two host membranes (the outer cell membrane and an inner vacuole membrane). Although intracellular plasmodia are very well protected from the host's immune response early in their growth, this strategy does create physiologic problems for the parasite. For example, the parasite must obtain its nutrients for growth through three membranes (two host and one parasite), and must eliminate its waste products through the same three membranes. Plasmodia solve this problem by appropriately modifying the host cell membranes. Parasitic proteins are incorporated into the red blood cell outer membrane. The host eventually responds to these antigens, and this response ultimately leads to the increased removal of infected host cells.

The existence of extracellular phases in the malaria life cycle is important, since immunization against these stages is the rationale for the development of our current vaccine candidates. The protective antigens on these extracellular stages have been purified as potential antigens for a vaccine. However, this approach has problems. For example, the sporozoite stage is exposed to protective antibody for only a brief period, and even a single sporozoite that escapes immune elimination will lead to an infection. Second, the antigenic variability of different isolates and the ability of different strains to undergo antigenic variation are not fully known. Therefore, the effectiveness of the vaccine candidates must still be demonstrated. However a large synthetic peptide containing antigenic sequences from 3 different proteins of P falciparum has been shown to reduce the clinical incidence of malaria by 31% in field trials. There is therefore optimism that a vaccine against P falciparum may be available in the near future.

A number of parasitic protozoa reside in macrophages. Although these organisms are protected from external immune threats, they must still evade digestion by the macrophage. Three strategies have been suggested. First, the parasite may prevent the fusion of lysosomes with the phagocytic vacuole. The actual mechanism responsible for this inhibition is not yet understood, but it has been shown to occur in cells infected with Toxoplasma. A second mechanism is represented by the ability of T cruzi to escape from the phagocytic vacuole into the cytoplasm of the macrophage. Finally, it is possible that some parasites can survive in the presence of lysosomal enzymes, as can the leprosy bacillus. One of the best-studied examples of a protozoan parasite able to survive in the phagolysosome is Leishmania. It has been suggested that the resistance of this parasite to the host's hydrolytic enzymes is due to surface components that inhibit the host's enzymes and/or to the presence of parasitic enzymes that hydrolyze the host's enzymes. As previously noted, at least one protozoan parasite, Theilera, is capable of growing directly in lymphocytes. Therefore, this parasite may escape the host's immune response by growing inside the very cells required for the response.

Antigenic Variation

Three major groups of parasitic protozoa are known to be able to change the antigenic properties of their surface coat. The African trypanosomes can completely replace the antigens in their glycocalyx each time the host exhibits a new humoral response. These alterations in serotype are one important way in which the African trypanosomes escape their host's defense mechanism. Although less well-characterized, similar changes are reported to occur in Plasmodium, Babesia, and Giardia.

It has been estimated that African trypanosomes have approximately 1,000 different genes coding for surface antigens. These genes are located on various chromosomes; however, to be expressed, the gene must be located at the end of a chromosome (telomeric site). The rate at which variation occurs in a tsetse-fly-transmitted population appears quite high. It has been shown that 1 in 10 cells appears to be capable of switching its surface antigen. The order in which the surface coat genes are expressed is not predictable. Much information is available on the nucleotide sequence of the genes coding the coat proteins; however, neither the factor(s) that induces a cell to switch its surface antigens nor the specific genetic mechanism(s) involved in the switch are fully understood. The antibody response does not induce the genetic switch, but merely selects variants with new surface antigens out of the original population. Considerably less information is available on the phenomenon of antigenic variation in malaria or babesiosis. However, antigen variation could be a major problem in reference to the development of a blood stage (merozoite) vaccine for malaria. Finally, antigenic variation has been observed in Giardia lamblia. A number of different gene families coding for surface proteins in Giardia have been identified. Antigenic variation has been suggested to assist Giardia in escaping the host's immune response.

Immunosuppression

Immunosuppression of the host has been observed with almost every parasitic organism carefully examined to date. In some cases the suppression is specific, involving only the host's response to the parasite. In other cases the suppression is much more general, involving the response to various heterologous and nonparasite antigens. It has not yet been proven that this immunosuppression allows the parasites to survive in a normally immunocompetent host. However, one can postulate that immunosuppression could permit a small number of parasites to escape immune surveillance, thus favoring establishment of a chronic infection. This mechanism might be particularly effective in parasites thai undergo antigenic variation, since it could allow the small number of parasites with new surface antigens to go undetected initially. Immunosuppression experimentally induced by various extraneous agents has certainly been shown to produce higher parasitemias, higher infection rates, or both. Therefore, the hypothesis that parasite-induced immmosuppression increases the chance for a parasite to complete its life cycle makes sense.

It should be noted that immunosuppression can be pathogenic itself. A reduced response to heterologous antigens could favor secondary infections. Humans suffering from malaria or trypanosomiasis have been shown to be immunosuppressed to a variety of heterologous antigens. Secondary infections may often be involved in death from African trypanosomiasis.

A variety of mechanisms have been suggested to explain the immunosuppression observed in protozoan infections. The most common mechanisms proposed are (1) the presence in the infected host of parasite or host substances that nonspecifically stimulate the growth of antibody-producing B cells, rather than stimulating the proliferation of specific antiparasite B-cells; (2) proliferation of suppressor T-cells and/or macrophages that inhibit the immune system by excretion of regulatory cytokines; and (3) production by the parasite of specific immune suppressor substances.

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