BACKGROUND: THE IMMUNE SYSTEM AND PARASITIC EVASION
Throughout the course of evolution, parasitic organisms have developed several methods to alter their environments so that they may survive and reproduce, thereby ensuring their propagation. These adaptive strategies can be passive or may involve active manipulation of the host's immune system through evasion, exploitation, and molecular piracy. The particular mechanisms used depend on the parasite’s:
Using Leishmania as our model parasite, we will discuss several such paradigms of parasitic immune evasion. Specifically, we will compare the intracellular evasion mechanisms of inhibition of phagosome-lysosome fusion, expression of MHC-I and II molecules, and peptide loading along with prevention of apoptosis to the extracellular mechanism of complement lysis evasion.
The host's mechanisms of defense against parasites range from the relatively simple primary barrier to more elaborate mechanisms that involve a variety of cells and molecules capable of specific recognition and elimination of pathogens (6). Following penetration of a physical barrier (the body's first layer of protection), the innate immune system generates an immediate but non-specific response. This can include the mobilization of immune cells by the production of cytokines; activation of the complement cascade to identify the invader, activate immune cells, and promote the clearance of dead cells and antibody complexes; identification and removal by white blood cells; and, if unsuccessful in eliminating the pathogen, activation of the adaptive immune system via antigen presentation. In the adaptive immune response, the pathogen is identified as non-self during antigen presentation, and this generates specific responses that are tailored to maximally eliminate the pathogen itself or cells it has infected. This results in the development of immunological memory so that future attacks are met with a stronger, faster immune response (7).
Figure 4: The two branches of the adaptive immune system: humoral (mediated by B lymphocyte production of antibodies) and cellular (mediated by T lymphocytes)
As shown in Figure 4, the adaptive immune response can be further broken down into cell-mediated and humoral components. Cell-mediated immunity involves the activation of macrophages and natural killer cells (destroy pathogens), antigen-specific cytotoxic T-lymphocytes (lyse infected cells), and the release of cytokines (influence functions of other cells). The humoral response refers to the production of antibodies by B-cells and activation of the accessory processes that accompany it: Th2 activation (T-helper cells), cytokine production, memory cell generation, and complement system activation. Extracellular parasites initiate the humoral immune response while intracellular parasites are susceptible to attack by the cellular response (6).
EXTRACELLULAR EVASION OF THE HUMORAL IMMUNE RESPONSE
The extracellular portion of the Leishmania life cycle focuses on the transition from the promastigote stage (existing in the sandfly vector and outside of the host macrophage) to the amastigote stage (existing inside the host macrophage) (8). After entering the human host during the blood meal of a female sandfly, Leishmania promastigotes must first evade complement-mediated lysis in the bloodstream until they are engulfed by a macrophage (9). The promastigotes are considered
part of the extracellular phase of Leishmania because they have not yet developed into intracellular amastigotes. At this point, the parasite is exposed to the potentially lethal effects of the complement system.
In normal humoral immune system functioning, the complement system functions to pierce the cell membrane of a pathogen, leading to its lysis. It consists of the membrane attack complex (MAC) (Figure 5), which is made up of complement proteins. (Each protein begins with C followed by a number reflecting their order of discovery.) The MAC must first be assembled and activated before it can attack invaders, and this can be done in one of two ways—the classical pathway or the alternative pathway. In each pathway, complement proteins are activated by successive cleavages and ultimately result in the formation of the MAC. The MAC is composed of C5b-8 and acts as a receptor for membrane-disrupting C9 molecules. When C5b-8 and poly-C9 bind, the MAC forms a transmembrane pore that leads to the lysis of the target cell (10).
In classical activation, complement proteins are activated by antibodies. Conversely, in the alternative pathway a variety of antigens and components of viruses or other pathogens activate complement proteins. Following complement pathway activation via the alternative pathway, MHC is also then able to insert into a pathogen’s cellular membrane and destroy it. Whichever pathway is used to activate the MAC, this system is essential for fighting off foreign invaders.
Click here to watch activation of the alternative complement pathway. For a more comprehensive explanation of the complement system, refer to (10).
Figure 6 compares the classical and alternative pathways.
Figure 6: The classical and alternative pathways both lead to cell lysisThe Leishmania parasite has devised a way to evade complement lysis so that it may go on and infect macrophages. Several hypotheses have been suggested to explain the exact mechanism. In general, each proposed mechanism prevents the proper functioning of the MAC complex, albeit at different points in the activation cascade.
Most research supports the theory that lysis evasion depends on the differentiation of promastigotes into metacyclic forms in the sandfly, which are resistant to complement. However, this mechanism is specific to the Leishmania major species, where procyclic promastigotes are unable to resist complement action.
Furthermore, when insect-stage procyclic promastigotes develop into infective metacyclic promastigote forms (Figure 4), their membrane is altered to prevent insertion. The major surface molecule of the promastigote, the lipophosphoglycan (LPG) is expressed on the parasite surface at this time and is considered a possible barrier for the insertion of the MAC C5b-C9 subunits into the parasite surface membrane because it is approximately twice as long as the form on procyclic promastigotes (11).
Alternatively, development into the metacyclic form is associated with changes in membrane carbohydrates, altered motility, and enzyme activities (12). The metacyclic forms are thus highly active and are larger in size because they have more protein and less carbohydrate, which could explain why it differs from the procyclic promastigote in complement reaction.
A different mechanism specific to Leishmania donovani promastigotes is the prevention of C5 convertase formation by fixing the inactive C3bi subunit on their surfaces (9). The surface glycoprotein gp63, a protease, has been reported to protect Leishmania amazonensis and Leishmania major against cellular lysis by converting C3b into C3bi, thus favoring parasite opsonization. Opsonization makes the parasite more susceptible to phagocytosis and thus less vulnerable to complement (9).
Interestingly, Leishmania also exploits the complement system to increase its infectivity of host cells. It uses complement activation as a means to be phagocytized by a macrophage. Unlike other parasites such as Toxoplasma gondii, Leishmania does not use a specific receptor on the host cell to gain entry and is instead passively taken up by macrophages. It relies on complement proteins and antibody to coat its surface, which then leads to opsonization—the ability of macrophages to take up opsonized (antibody-coated) particles (7). By subverting the complement system and exploiting it as a means for host cell entrance, Leishmania is able to persist within its extracellular environment.
As mentioned earlier, Leishmania lives primarily in macrophages (Figure 7). Macrophages are derived from white blood cells and are found in the tissues. Their role is to clear cellular debris and pathogens from the body through phagocytosis. In normal cellular immune system functioning, the macrophage ingests a parasite into a phagosome (Figure 8), which fuses to one of the cell's many late endosomes and lysosomes. Parasite proteins are then degraded into short peptide fragments, which the macrophage then presents in the context of MHC class II molecules to CD4+ helper T cells, another type of white blood cell, in the lymph node. Macrophages may also display parasite peptides in the context of MHC class I molecules to CD8+ cytotoxic T cells. This activates the cytotoxic T cells to proliferate and destroy the infected macrophage through the secretion of the cytokine interferon gamma (IFN-g) (7).
Figure 7: A macrophage
Figure 8: Ingestion of a Leishmania parasite by a macrophage
Alan Sher of the National Institute of Health notes that "it's kind of ironic that Leishmania decides to live in the most dangerous cell in the body” (3). It is only able to do so, however, by manipulating the macrophage's machinery in various ways. Most notably, Leishmania inhibits the macrophage proteolytic process by preventing the fusion of its own parasitophorous vacuole (a nonfusigenic version of a phagosome that lacks important host surface proteins) with the cell's lysosomes (Figure 9). LPG, which has been previously mentioned in the extracellular evasion of complement, is imperative in this process as well. Studies have confirmed that the LPG surface protein becomes incorporated into the phagosome membrane and somehow inhibits parasitophorous vacuole fusion with endosomes, in spite of the fact that the host cell fusion machinery remains operational during infection (14). The exact mechanism by which LPG prevents phagosome-endosome fusion is not known.
Figure 9: Parasitophoric vacuoles inside a host cell containing 2 or more parasites
LPG is also implicated in the prevention of apoptosis by Leishmania-infected macrophages. Apoptosis is a signal-dependent physiologic suicide mechanism that allows for the homeostatic balance between cell proliferation and cell death (Figure 10). It involves the transcription of specific genes and requires a trigger from the environment, usually exposure to or the removal of a specific growth factor or hormone. It has been shown that LPG inhibits apoptosis in bone marrow macrophages in vitro and that the parasite also induces the expression of a number of cytokine genes (15). Therefore it is thought that with the help of LPG, Leishmania-mediated inhibition of apoptosis may be related to the stimulation of cellular cytokine gene expression. Specifically, the cytokine TNF a is induced in infected cells, and acts in an autocrine manner to inhibit apoptosis with the help of additional factors such as LPG. However, if the phagolysosome does form, another parasitic surface protein, gp63, inhibits degradative phagolysosomal enzymes (16). This also ensures the survival of the parasite within the host cell.
Figure 10: Comparison of a normal WBC to an apoptotic WBC
Lastly, Leishmania promotes its survival within the macrophage by down regulating MHC molecules and by preventing their antigen presentation. The glycosylinositolphospholipid Leishmania component has been found to prevent the expression of both classes of MHC molecules. The degree of gene suppression correlates with both the duration of infection and the parasitic load (17). This is supported by the fact that increasing quantities of IFN-g do not remedy this. (Recall that IFN-g stimulates infected macrophages to die.) For the few MHC molecules that are produced, however, the parasite is able to prevent peptide loading (Figure 11). Gp63 cleaves the CD4+ molecules on T cells, undermining the interaction between antigen-presenting cells and T helper cells (18). One hypothesized mechanism involves the release of proteolytic enzymes by the parasite in the vicinity of peptide loading onto the MHC molecules, essentially degrading the peptides "on the spot" (19). Thus, Leishmania is capable of subverting key macrophage accessory functions that are required for the induction of T cell-dependent anti-parasite immunity.
Figure 11: MHC class I and II peptide loading