Avoidance of Innate Immune Mechanisms by the Protozoan Parasite, Leishmania spp

Mosser DM, Miles SA.

Publication Details

In this chapter, we will examine the mechanisms by which Leishmania parasites interact with host cells. We will try to develop the hypothesis that the success or failure of Leishmania infections can be traced to the initial mechanism(s) of parasite entry into mononuclear phagocytes. We will try to make the following points about the activation and modulation of innate immunity by Leishmania: First, promastigotes enter macrophages by a quiescent mechanism that fails to induce innate immune responses, and this may result in a delayed induction of an adaptive immune response. This delay in the development of adaptive immunity may provide the parasite with time to replicate within macrophages. Second, parasite replication disrupts macrophage responsiveness to the immune signals that are eventually generated. Third, the mechanism of amastigote entry into macrophages may also be the harbinger for successful parasitism. Amastigotes coat themselves in host IgG which ligates macrophage FcγR, resulting in the hyperproduction of IL-10 from infected macrophages. This IL-10 can prevent macrophage responses to IFN-γ allowing the parasites to survive even in the immunologically intact host.

Life Cycle

The promastigote form resides in the midgut of infected sandflies (Fig. 1). A small pool of blood is formed when a female fly takes a blood meal. Infectious promastigotes (metacyclics) are regurgitated into this pool of blood, where they enter the human host.1 Phagocytic cells recruited to the site of infection rapidly internalize the promastigotes. The parasites not only survive but replicate within the acidic phagolysosome. Replication continues until the host cell is lysed, releasing parasites, to infect neighboring cells.

Figure 1. The life cycle of Leishmania spp.

Figure 1

The life cycle of Leishmania spp. The promastigote stage resides in the midgut of sandflies, and is transferred to the human host during a bloodmeal. Promastigotes are taken up by resident macrophages and cells recruited to the site of the bite. Inside (more...)

Receptor Mediated Phagocytosis

The uptake of Leishmania promastigotes by macrophages is a receptor-mediated process that involves the expenditure of energy by the macrophage, but not by the parasite.2 Due to the obligate intracellular nature of the pathogen, this organism expresses several different ligands on their surface that can interact with a variety of different macrophage receptors, to ensure their uptake by phagocytic cells.2 These include the receptors for complement,3,4 fibronectin,5 and sugars, such as the mannose-fucose receptor6 and others.7 These receptors bind parasites with different avidity. In some instances low affinity receptors can make a substantial contribution to parasite internalization without making an obvious contribution to parasite adhesion. A clear example of this is the fibronectin receptor (FnR), which binds to the parasite surface molecule gp63 with low affinity. During in vitro phagocytosis assays, this receptor appears to play little or no role in parasite adhesion. However cells lacking FnR, or parasites with a mutation in the fibronectin recognition domain on gp63, exhibit significant delays in parasite uptake, relative to wild-type cells,6 indicating that this receptor plays an important role in uptake. The ligands on promastigotes that have been implicated in parasite uptake include lipophosphoglycan (LPG) and gp63, as well as other phosphoglycan species in the parasite glycocalxy.8-10 However the most important ligands for parasite uptake are not parasite-encoded at all. Promastigotes rely heavily on host-derived opsonins to achieve maximal uptake by phagocytis cells. In addition to fibronectin, mentioned above, the complement system represents an important mediator of promastigote adhesions to phagocytic cells.11 The third component of complement (C3) and the complement receptor Type 3, (CR3, Mac-1, CD11b/CD18) is probably the most important of the macrophage complement receptors for parasite phagocytosis. This is due to the abundance of CR3 expression on macrophages and to the very transient nature of the C3b molecule, whose half-life on opsonized particles is measured in minutes.12

Promastigotes enter into macrophage phagosomes, which acidify and fuse with lysosomes. It is in these acidified phagolysosomes that the organisms replicate. The evidence to indicate that these phagolysosomes are fully competent, is the fact that debris and dead organisms are degraded in the same phagolysosomes that house viable amastigotes.13 There is now strong evidence that there is a delay in the maturation of promastigote phagosomes. This “pregnant pause”14 may give the promastigote the time needed to transform into amastigotes and upregulate the genes necessary for intracellular survival.15 Lipophosphoglycan from the surface of the promastigote may contribute to this delay in maturation. The current thought is that alterations in the lipid content of cellular organelles can influence the fusagenic competence of these vesicles with each other.16 The intercalation of LPG into the membrane of the phagosome may adversely influence this fusion process, delaying maturation long enough for parasite survival.17 This delay may not be exclusively mediated by LPG, since some LPG-deficient species, such as L. mexicana, survive quite well in macrophages.18

Amastigotes are also taken up by receptor-mediated phagocytosis and this process is remarkably efficient. A resting macrophage can internalize a dozen or more amastigotes within 30 minutes. While this developmental form may use some of the same receptors as the promastigote form, there are clearly some important differences with regard to amastigote uptake. Amastigotes appear to have a heparin binding activity, which allows them to adhere to cellular proteoglycans. This adhesion increases the efficiency of receptor-mediated phagocytosis. Mannose receptors and complement receptors have been implicated in amastigote phagocytosis,5 and the amastigote ligands include phosphoglycans19 and glycoinositol-phospholipids (GIPL).20 Interestingly, a study has shown that amastigotes may mimic apoptotic cells and bind to phosphatidylserine receptors on macrophages.21-23 This would be consistent with the failure of these organisms to activate inflammatory cytokine production from macrophages. Similar to promastigotes, a host opsonin plays an important role in amastigote uptake. The opsonin in this case is IgG, rather than complement. Amastigotes isolated from lesions of experimentally infected animals are coated with host IgG and they bind avidly to macrophage Fcγ receptors (FcγR).5 It is important to note that like the promastigote, there are multiple, redundant ways for amastigotes to enter macrophages. Consequently, neither the lack of IgG on amastigotes, nor the lack of FcγR on macrophages, prevents amastigote phagocytosis. The role of IgG on amastigotes will be discussed in detail in a subsequent section.

Amastigote entry into macrophages does not result in a delay in phagolysosomal (P/L) fusion.24 It would be interesting to know whether signals transduced through the FcγR are responsible for the efficient uptake of amastigotes leading to P/L fusion. It should also be noted that dendritic cells express FcγR and are highly phagocytic for both developmental forms of Leishmania. There is solid evidence that signaling through FcγR leads to DC maturation25-27 and enhanced APC function. The role of FcγR-mediated phagocytosis of Leishmania by DCs may represent an important mechanism for the induction of a protective immune response.28

Leishmania and Innate Immunity

There is a growing body of evidence to suggest that the activation of the innate immune response occurs during experimental infection with Leishmania, and that this activation can impact the development and the characteristics of the adaptive immune response. Mice on a resistant background, but deficient in the toll-like receptor (TLR) adaptor MyD88, developed progressive lesions and failed to mount protective Th1 immune responses.29,30 Similarly, genetically resistant mice, which carried a null mutation in tlr4, developed non healing lesions, when infected with L. major.31,32 There is also evidence that parasite derived molecules can activate TLRs. The most thoroughly studied of these is LPG, which has been shown to activate TLR 2.30 There is evidence from other protozoa that glycosyl-phosphatidylinositol (GPI) anchored structures can activate TLRs,33 although this may not be true for at least some GPI-linked structures from Leishmania.34

Despite these in vivo studies, which suggest a role for innate immunity, the results from in vitro infections point to fundamental differences in the innate response of macrophages and dendritic cells to this organism, relative to bacteria. The phagocytosis of even small numbers of bacteria is invariably associated with the rapid translocation of NF-κB,35 and the secretion of a large array of cytokines, most of them inflammatory in character, such as TNF, IL-1, and IL-12. This rapid activation of innate immune responses does not appear to occur following Leishmania infection. Even the phagocytosis of large numbers of promastigotes results in little, to no, detectible NF-κB activation (Fig. 2). Consequently, promastigote phagocytosis induces minimal cytokine secretion by infected macrophages (Fig. 3). This appears to be a characteristic of all Leishmania species tested, regardless of whether they are alive or heat-killed, provided that the organisms are grown in media that is free of contaminating LPS. Thus, the promastigote form of the parasite enters macrophages and dendritic cells by a quiescent mechanism that does not elicit substantial cytokine production. Importantly, the simultaneous addition of LPS and parasites to macrophages results in the secretion of a large number of LPS-induced cytokines. The only exception of which is IL-12 which will be discussed below. The logical interpretation of this latter observation is that Leishmania promastigotes do not actively prevent cytokine production by macrophages, but rather they fail to induce it. The easiest way to reconcile the in vivo observations suggesting that TLRs are an important component of early immunity to Leishmania, and the in vitro evidence that parasites induce minimal TLR activation, is to hypothesize that during infection, the indirect activation of endogenous TLR ligands by Leishmania, such as those associated with the inflammatory extracellular matrix, are responsible for the induction of innate immunity. This indirect activation of innate immunity may account for the slow induction of immune responses that occurs when mice are infected with low numbers of parasites in the ear.36

Figure 2. Immunofluorescence microscopy to detect NF-κB (p65) translocation in macrophages.

Figure 2

Immunofluorescence microscopy to detect NF-κB (p65) translocation in macrophages. Untreated macrophages show cytoplasmic staining for NF-κB (p65) and dark unstained nuclei. LPS-treated macrophages show brightly stained nuclei indicating (more...)

Figure 3. TNF production by infected macrophages.

Figure 3

TNF production by infected macrophages. Unprimed (black bars) or IFN-γ-primed (gray bars) macrophages were infected with increasing ratios of live or heat-killed promastigotes of L. amazonensis. Twenty-four hours later, TNF levels in supernatants (more...)

Suppression of IL-12 and the Th1 Response

The failure of Leishmania promastigotes to induce the secretion of macrophage cytokines extends to IL-12.37,38 However, in the case of IL-12, promastigotes not only fail to induce IL-12 synthesis, but they appear to actively downregulate its production (Fig. 4).39 This is different from other inflammatory cytokines, which are not actively suppressed by Leishmania infection. The in vitro infection of macrophages, with either developmental form of the parasite, results in a general suppression of IL-12 biosynthesis, even in response to exogenous stimuli, such as LPS. This observation is quite unexpected, given the importance of IL-12 in directing the development of a protective Th1 immune response.40 The downregulation of IL-12 leads to a failure in macrophage activation, and ultimately a failure in the intracellular killing of Leishmania. This inhibition may be a general response to infection, or it may be a result of the increased levels of intracellular calcium that seem to accumulate in infected macrophages (see below).41-43 Alternatively, it has been suggested that the specific suppression of IL-12 may relate to the transcription factor IFN-γ consensus sequence binding protein (ICSBP). Activation of the IL-12(p40) promoter requires ICSBP, which is regulated by signal transducer and activator of transcription 1 (STAT-1).44 Thus STAT-1 independent pathways may allow other cytokines to be produced, and remain unaffected by the presence of Leishmania. Similar to intact parasites, the stimulation of macrophages with purified LPG also diminishes the production of IL-12 by macrophages. It is likely, however, that this response is not specific to LPG, because the ligation of a large number of different receptors on macrophages has been associated with a decrease in IL-12 biosynthesis.45-47 Regardless of the mechanism, the dramatic inhibition of IL-12 following Leishmania infection points to other nonmacrophage sources of IL-12 as being responsible for the initiation of the protective Th1 biasing that occurs during a protective immune response to this parasite.

Figure 4. IL-12 production by infected macrophages.

Figure 4

IL-12 production by infected macrophages. IL-12 production by macrophages was measured by ELISA 24 hrs after exposure of cells to 10 μg/ml LPS alone or in combination with a 10:1 ratio of L. amazonensis promastigotes.

Alterations in Signal Transduction following Leishmania Infection

In addition to failing to efficiently activate innate immunity in parasitized cells, it also appears as though promastigote infection of macrophages is associated with alterations in cellular signal transduction mechanisms, leading to defective immune responses of infected macrophages. Thus, the parasite may take an active role in suppressing immune responses. The experimental model used for most of these studies is to infect macrophages with Leishmania, and then look for alterations in signal transduction by infected cells. Obviously in a model such as this, the multiplicity of infection is quite important, and to our knowledge, a careful study to correlate signaling alterations with MOI values has not been done. Nevertheless, several observations have begun to emerge about specific alterations in signaling in parasitized macrophages.

One of the early observations, which has been confirmed by several groups, is that the infection of macrophages with Leishmania is accompanied by an increase in intracellular calcium levels.41 This may have several consequences on parasite survival. Alterations in the activation of various isoforms of protein kinase C (PKC)48,49 have been reported in infected macrophages, and these alterations may be causally related to the changes in intracellular calcium concentrations. The experimental inhibition of PKC activation in macrophages leads to increased numbers of parasites within these macrophages.49 Additionally, it has been shown that purified LPG can block PKC activity, and that promastigotes which do not express LPG on their surfaces exhibit decreased intracellular survival.50 These observations suggest that parasite induced impairment of PKC activation may be important mechanism of establishing an infection. Altered levels of intracellular calcium may also be involved in the reported alterations in mitogen-activated protein kinase (MAPK) activation that accompany Leishmania infection.51,52 MAPKs play an important role in promoting the transcription of many inflammatory cytokines,53,54 and the differential activation of MAPKs has been linked to the production of different cytokines during infection. Recent evidence suggests that activation of different MAPKs can influence parasite survival.55 The suppression of IL-12 production during infection may be a result of the preferential activation of extracellular signal related kinase (ERK) 1/2, by Leishmania.56

The strongest evidence for alterations in signal transduction pertain to the inhibition of protein tyrosine kinase (PTK) activity in infected macrophages. This observation has been made by several groups.57-59 The reduction of PTK activity in infected macrophages leads to reduced signaling through Janus kinases (JAKs) and STAT-1.60 The major result of this alteration is defective responses of infected macrophages to the activating effects of IFN-γ.61 Infected macrophages display reduced levels of total and phosphorylated JAK1 and JAK2.62 Additionally, STAT-1 plays a critical role in the signaling of IFN-γ. The STAT-1 mediated pathway has been shown to be necessary for a protective immune response against L. major.63 L. donovani has been shown to attenuate IFN-γ induced STAT-1 phosphorylation in macrophages, aiding the parasite in escaping host immunity.60 The mechanism whereby Leishmania modulate cellular PTK activity appears to involve the activation of cellular phosphatases.64 Cellular SHP-1 activity is increased following infection, and subsequent stimulation of macrophages with phorbol esters to stimulate protein tyrosine phosphorylation results in diminished levels of tryosine phosphorylation in infected cells.65 The mechanism of SHP-1 activation by Leishmania has not been defined, but given the sensitivity of these molecules to alterations in lipid composition, one cannot rule out a role for the inositol-linked phospholipids from the Leishmania surface, which appear to intercalate into the plasma membrane of infected macrophages.66

IL-10 Induction by IgG-Opsonized Amastigotes

It is well-established that the infection of BALB/c mice with L. major results in the production of IL-4 from T cells, which ultimately results in progressive disease. However, there is increasing evidence that IL-4 may not be sufficient for susceptibility of BALB/c mice to cutaneous leishmaniasis.67,68 Early studies in human visceral leishmaniasis correlated IL-10 levels with disease severity,69 and several groups have subsequently examined the role of this cytokine in murine models of disease. The role of IL-10 in animal models of leishmaniasis has been demonstrated for both cutaneous and visceral forms of the disease.38,67,70 This is important, because IL-10 can suppress Th1 responses, and inhibit macrophage activation.71

It has been previously demonstrated that macrophages activated in the presence of immune complexes shut off their production of IL-12,45 and produce high levels of IL-10.72 This alteration in cytokine production occurs when immune complexes ligate the FcγRs on macrophages. The mechanisms involved in this cytokine switch are not fully understood, however it has been shown that ERK activation is required for the super-induction of IL-10 caused by immune complexes.73 FcγR ligation leads to the activation of the ERK, which leads to remodeling of the il-10 promoter, making it more accessible to transcription factors that bind there.73 The high level of IL-10 production from macrophages has two important effects. First, IL-10 makes macrophages refractory to the activating effects of IFN-γ.38 Second, when macrophages are used as antigen presenting cells, the production of IL-10 inhibits the development of Th1 cells and induces a Th2-like immune response.74

These basic observations predicted that the presence of immune complexes could adversely influence the development of cell mediated immunity to Leishmania spp. Two other previous observations reinforced this prediction. The first was the observation that the levels of immune complexes are quite high in patients with visceral leishmaniasis.75,76 The second was the observation that amastigotes isolated from the lesions of infected mice were opsonized with host IgG.38,77 Thus, immune complexes formed during Leishmania infections could interfere with protective immune responses to this organism.

A series of studies was undertaken to determine whether lesion-derived amastigotes could induce IL-10 production from macrophages in an FcγR-dependent manner. In both murine and human systems this is precisely what we observed. Lesion derived amastigotes from BALB/ c mice induced high levels of IL-10 production from inflammatory macrophages expressing FcγR.38 Similarly, peripheral blood mononuclear cells from the blood of normal human donors produced high levels of IL-10 when infected with L. chagasi amastigotes that were opsonized with serum from a patient with visceral leishmaniasis (VL).78 In vivo studies in JH mice were consistent with these in vitro observations. JH mice have a targeted deletion of the immunoglobulin heavy chain J locus, and therefore, make no antibody.79 JH mice were more resistant to infections with L. major than were normal BALB/c mice. When these mice were reconstituted with sera from chronically infected BALB/c mice, they exhibited larger lesions, with higher numbers of parasites residing in those lesions. Treating the mice with a mAb to the IL-10 receptor prevented the IgG-mediated enhancement in lesion development.78 These studies demonstrate that host IgG can cause a novel form of immune enhancement, due to its ability to induce IL-10 production from macrophages. These studies also provided evidence to link the high titers of parasite-specific IgG in human VL with defective DTH responses and disease progression.

These observations point to several interesting parallels between L. major infection of BALB/c mice, and human VL. BALB/c mice develop high antibody titers, with frequent metastasis of parasites to the bone marrow, liver, and spleen.80 Additionally, the delayed-type hypersensitivity (DTH) response in BALB/c mice is smaller and more transient than in resistant C56BL/6 mice.81 Interestingly, 20 years ago it was demonstrated that the suppression of DTH responses in BALB/c mice required the presence of B cells.82 Taken together these observations suggest that parasite-specific antibody can play a role in disease by contributing to the formation of immune complexes, which in turn induce the production of IL-10 from host macrophages. These conclusions are consistent with previous reports by others,5,78,83,84 which demonstrated that B cell deficient mice, on the susceptible BALB/c background, were more resistant to disease, and developed smaller lesions. It is important to note that in addition to macrophages,85 Th2 and T regulatory cells (Tregs) have also been shown to be important producers of IL-10. The role of Tregs was specifically examined in mice infected with low doses of L. major.86 It was shown that IL-10 derived from Tregs made an important contribution to the persistence of parasites after clinical cure. Paradoxically, the sterile cure observed in mice deficient in IL-10 was actually detrimental to host immunity, because these mice were no longer immune to subsequent reinfection following clearance.87 This work indicated that the maintainance of memory T cells is dependant upon parasite persistence. The mechanism of Treg induction by these parasites is not known. However, there appear to be multiple ways that this organism induces an IL-10 response from the host that is conducive to parasite growth and/or persistence. The IL-10 produced by macrophages and Tregs appears to cause a localized and transient immunosuppression that allows the parasite to survive in an immunocompetent host.

Conclusion

Parasites in the genus Leishmania have evolved a variety of intricate ways to modulate the host immune response. The promastigote relies on a stealth mechanism of entry to parasitize macrophages without inducing the panoply of cytokines that designates “danger” to the host. Furthermore, the intracellular residence of this parasite in phagocytes confuses the signaling pathways, making these cells less efficient at responding to immune activating signals. Finally, the amastigote form of the parasite takes advantage of what should normally be a protective immune response, and coats itself in host IgG, in order to induce the production of the immunosuppressive cytokine, IL-10.

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