Macrophages serve either as host and primary effector cells against Trypanosoma cruzi, the protozoan parasite responsible for Chagas disease. Although the parasite mobilizes innate and adaptive immune responses that induce macrophage activation and keep infection under control, T. cruzi persists in the host for life. Parasite persistence is associated with inflammatory destruction of skeletal and cardiac muscle. Here, the roles of T. cruzi molecules and immune mechanisms involved in parasite evasion of macrophage defenses are discussed. Targeting these molecules and mechanisms will be essential for attaining successful therapies and vaccination.

Introduction

Macrophages play important roles in Trypanosoma cruzi infection by serving either as host and effector cells against the parasite. Macrophages are among the first cells to be parasitized by T. cruzi.¹ Early studies demonstrated that T. cruzi multiplies in resident macrophages, leading to rupture of the host cell, and to the release of infective trypomastigote forms.^2,3 However, it was soon apparent that macrophages could be instructed by the host immune response to become effector cells against the parasite. One central observation was that previous activation of macrophages by products released from activated lymphocytes - later identified as proinflammatory cytokines, including IFN-γ-induced microbicidal activity, conferring the ability to control infection.^2,3 Macrophages play a protective role in T. cruzi infection in vivo. Macrophage depletion by treatment with silica increases parasitemia and rates of mortality in infected mice.⁴ However, macrophages could play a deleterious role, as well. Depletion of macrophages by treatment with clodronate liposomes resulted in decreased parasitism and damage to the central nervous system in suckling rats,⁵ suggesting that macrophages might serve to disseminate infection. These contrasting roles of macrophages could be explained by induction of functionally diverse pathways of macrophage activation.

Distinct Programs of Macrophage Activation

Microbial products acting in concert with cytokines secreted by T-cell subsets influence the functional outcome of effector macrophage differentiation. At least three distinct phenotypes have been identified. The best characterized cells are the classically activated macrophages (caMφ), which are the effector cells of Th1 immune responses.⁶ caMφ differentiate in the presence of two signals. One is IFN-γ, a major cytokine secreted by Th1 CD4⁺ T cells and CD8⁺ T cells. The second signal is TNF-α, which can be induced by microbial products acting as ligands for Toll-like receptors (TLRs). caMφ produce nitric oxide (NO), increase expression of class II MHC and costimulatory CD86 molecules, and have increased antigen presenting capacity. caMφ play an essential protective role against intracellular pathogens due to combined NO release and increased oxidative burst, as well as secretion of proinflammatory cytokines TNF-α, IL-1 and IL-6.⁷

Macrophages activated in a Th2 environment express different gene products, and were termed alternatively activated macrophages (aaMφ). In contrast to caMφ, aaMφ fail to generate NO from L-arginine. Macrophages upregulate arginase activity following interaction with Th2 cytokines IL-4 and IL-10,⁸ or with the immunossuppressive cytokine TGF-β.⁹ Arginase and the downstream enzyme ornithine decarboxylase (ODC) shift L-arginine metabolism towards production of polyamines, such as putrescine.¹⁰ Arginase activity is a marker of aaMφ.⁷ aaMφ also express increased class II MHC and CD86 expression, and efficiently present antigen to T cells.¹¹ However, aaMφ secrete antiinflammatory cytokines, such as IL-10 and TGF-β, and actively participate in reactions involving tissue remodelling, angiogenesis and wound repair during the healing phase of acute and chronic inflammatory diseases.⁷ Recently, it has been demonstrated that differentiation to caMφ requires the enzyme src homology 2-containing inositol-5' phosphatase (SHIP), a potent inhibitor of phosphatidylinositol 3-kinase (PI3-kinase).¹² Macrophages from SHIP-deficient mice differentiate into an aaMφ phenotype in vivo, which requires the action of active TGF-β present in the serum.¹² These studies will help understanding signaling pathways involved in distinct macrophage differentiation programs.

Macrophages activated by immune complexes through ligation of FcγRs in the presence of a microbial TLR ligand such as bacterial lipopolysaccharide (LPS), downregulate IL-12 secretion and produce increased amounts of IL-10.⁶ These cells preferentially induce Th2 responses characterized by IL-4 secretion and antibodies of the IgG1 isotype, and were called Type 2-activated Mφ because they are biased to induce a Th2 response.⁶ Type 2-activated Mφ comprise a distinct subset sharing some properties with both caMφ and aaMφ. Type 2-activated Mφ secrete TNF-α, IL-1 and IL-6, like caMφ. However, they do not secrete IL-12, they do secrete IL-10, and drive a Th2 response, like aaMφ. In contrast to aaMφ, type 2-activated Mφ do not express arginase activity.⁷ At present, it is unclear whether discrete activation states exist in vivo, or whether macrophages instead show a wide range of phenotypes, depending on multiple inflammatory stimuli.¹³ Monocyte and macrophage heterogeneity has been investigated by selective expression of cell surface markers, and this topic has been recently reviewed.¹³

Protective Mechanisms against T. cruzi Induced by IFN-γ

The ability of caMphi; to mediate microbicidal activity against intracellular forms of T. cruzi depends on priming with IFN-γ and requires production of NO.^14,15 Administration of IFN-γ reduced parasitemia and increased survival of mice infected with T. cruzi.¹⁶ Furthermore, mice receiving neutralizing antibody against IFN-γ, or deficient in IFN-γ Receptor had high parasitemias and succumbed from infection.^17,18 These results indicate a central role for IFN-γ in resistance against infection. IFN-γ is produced by effector Th1 CD4⁺ T cells and by CD8⁺ T cells upon antigen recognition, and activates transcription of a large number of genes in macrophages. Ligation of IFN-γ Receptor by IFN-γ induces nuclear translocation of STAT1 to stimulate the transcription of IFN-regulated genes. Several products of IFN-regulated genes inhibit pathogen survival.¹⁹ Indoleamine 2,3-dioxygenase (IDO), nitric oxide synthase 2 (NOS2), and phagocyte oxidase (Phox) inhibit the survival of intracellular bacteria and protozoa. NOS2 is located in the cytosol, where it catalyses the production of NO, which rapidly diffuses to the phagosome to effect pathogen killing. Phox is found in plasma membrane, cytosol and intracellular granules, but translocates to the phagosome, where it catalyses the conversion of O₂ to superoxide, which gives rise to molecules that are toxic to phagosome-bound pathogens. It has been suggested that NO combines with superoxide to generate peroxynitrite, which is more toxic to T. cruzi than NO.¹⁰ Several studies indicate that NO production by caMφ is a major mechanism of defense against T. cruzi. However, one recent study argued that NO-independent mechanisms triggered by IFN-γ are important for resistance against T. cruzi, specially in infections mediated by less virulent parasite isolates.²⁰ In this regard, IFN-γ induces expression of several GTP-binding proteins. A family of 47 kDa GTPases induced by IFN-γ has been implicated in intracellular defense against pathogens independently of NO.¹⁹ Although their mechanism of action is incompletely understood, p47 GTPases associate with lipid membranes and promote fusion of phagosomes harboring microbial pathogens with lysosomes.¹⁹ In addition, p47 GTPases regulate survival and function of memory-activated CD4⁺ T cells.¹⁹ Interestingly, IFN-γ induces autophagy, and the p47 GTPase LRG-47 is involved in autophagic killing of Mycobacteria by macrophages.²¹ p47 GTPases are important for resistance against intracellular parasites residing in phagosomes, such as Toxoplasma, Leishmania and Mycobacteria.¹⁹ Surprisingly, mice deficient in LRG-47 also express enhanced susceptibility to infection by T. cruzi,²² a pathogen that rapidly escapes the phagosome and enters the cytosol.¹ In this model, LRG-47 appears to be required for proper control of host lymphocyte numbers, and for NO-independent killing of T. cruzi induced in macrophages by IFN-γ.²² The mechanism of parasite killing promoted by LRG-47 remains to be identified. Other potential mechanisms of protection mediated by IFN-γ could involve antigen processing and presentation, and lymphocyte migration to infection sites.²⁰ These important issues also remain to be investigated.

Evasion of Innate Macrophage Defenses

Microscopic examination of T. cruzi replication in resident and inflammatory macrophages illustrates how permissive these cells are for the parasite. Studies performed with nonprofessional phagocytes indicate that T. cruzi invades host cells by a mechanism resembling membrane wound repair. This mechanism involves sustained increases in cytosolic Ca²⁺ levels, leading to recruitment of lysosomes to the site of parasite attachment.²³ The ability of parasites to trigger intracellular free Ca²⁺ transients in host cells is associated with the activity of a T. cruzi serine hydrolase, oligopeptidase B. Deletion of the gene encoding this enzyme results in a marked defect in host cell invasion and in the ability to infect mice.²⁴ Endocytosis of the parasite is achieved through fusion of lysosomes with the plasma membrane, followed by membrane recycling. Recent studies suggest the existence of a second route of parasite invasion independent of targeted lysosome exocytosis, provided by tightly associated plasma membrane derived vacuoles.²⁵ On the other hand, it has been suggested that T. cruzi invades professional phagocytes by a mechanism dependent on PI3-kinase and actin filament assembly, that resembles phagocytosis.²⁶ Interestingly, recent studies have suggested that the lysosome dependent pathway of invasion of nonphagocytic cells also requires PI3-kinase and host cell actin polymerization.^25,27 Both the parasite and the host cell rapidly initiate signaling pathways, allowing T. cruzi entry and intracellular survival. Similar to host cell signaling, penetration of T. cruzi into mammalian cells depends on the activation of the parasite protein tyrosine kinase activity and Ca²⁺ mobilization.²⁸ Later on, the parasitophorous vacuole is lysed under the action of a T. cruzi protein named TcTox, that resembles C9 complement component, and the parasite is released into the host cell cytoplasm.²⁹ Therefore, the parasite rapidly evades the hostile environment of the phagosome.³⁰ Compared with other pathogenic parasites, T. cruzi invades host cells silently, eliciting few changes in host cell transcription during the first hours of infection.³¹ Once in the cytoplasm, the parasite differentiates to amastigotes, replicates, and differentiates back to trypomastigotes. T. cruzi induces NF-kB activation in a number of cells which are relatively resistant to infection; but fails to do so in muscle cells, which are susceptible to invasion.³² It has been suggested that this cell type specific activation of NF-kB could explain the tissue tropism of T. cruzi.³² In addition, T. cruzi inhibits programmed cell death in certain mammalian cells, such as fibroblasts. The parasite posttranscriptionally upregulates expression of cellular FLICE inhibitory protein (c-FLIP); the only know mammalian inhibitor specific for death receptor signaling.³³ In this way, the parasite prolongs host cell survival, allowing more time for completing its replicative cycle.

Following rupture of the host cell, trypomastigotes are released to perpetuate the infective cycle. This infectious mechanism is facilitated by T. cruzi trans-sialidase,³⁴ and requires TGF-β signaling,³⁵ possibly to prevent harmful reactions of the host cell, and to provide essential cofactors and nutrients to the parasite. The exact nature of ligands and receptors critically involved in parasite invasion is unclear. However, invasion of host cells by T. cruzi appears to involve a complex array of host and parasite surface molecules. The extracellular matrix protein Fibronectin binds to T. cruzi trypomastigotes,³⁶ and facilitates infection of fibroblasts and macrophages.^36,37 In agreement, β1 integrins with avidity for Fibronectin are involved in infection.³⁸ Complement component C1q enhances invasion of phagocytes and fibroblasts by T. cruzi trypomastigotes opsonized wtih human serum.³⁹ Interestingly, C1q also bridges apoptotic cells to calreticulin-CD91 complexes on phagocytes, promoting engulfment of apoptotic cells.⁴⁰ Since engulfment of apoptotic cells inhibits proinflammatory properties of macrophages,⁴¹ it will be important to investigate whether trypomastigotes also engage calreticulin-CD91 complexes at the host cell. This strategy has been called “apoptotic mimicry” to indicate that parasites expose ligands similar to apoptotic cells in order to inactivate host macrophages and facilitate infection.⁴² The role of NO production at this early stage favors parasite infection. T. cruzi induces a modest and IFN-γ independent increase in NOS2 expression.43 Together with parasite NOS,¹⁰ parasitized cells produce low concentrations of NO, which facilitates proliferation of amastigotes.⁴³

Evasion of Activated Macrophages

Once a specific immune response has developed, caMφ which have differentiated through the IL-12/IFN-γ axis play a central role in control of parasitemia.⁴⁴ In fact, caMφ are not permissive to T. cruzi infection,² and parasite killing can be triggered in these cells by autocrine TNF-α secretion.^14,15 Microbicidal activity relies on large levels of NO production by highly expressed NOS2. Furthermore, in the presence of opsonizing antibodies, phagocytosis of T. cruzi engages FcγRs to stimulate peroxide production by Phox.^10,45 Peroxide reacts with NO to generate peroxynitrite in vitro.¹⁰ It has been suggested that the combined activation of NOS2 and Phox could lead to peroxynitrite formation to account for more efficient microbicidal activity of caMφ.¹⁰

Since infection by T. cruzi cannot be spontaneously cured, even in genetically resistant hosts, the parasite must be able to evade responses mounted by caMφ. Similar to other parasites that replicate in macrophages, T. cruzi has evolved molecular mechanisms to escape the microbicidal arsenal of the activated phagocyte,³⁰ or even to prevent phagocyte activation.⁴⁶ In fact, macrophages infected with T. cruzi have been observed in inflammatory lesions of chronically infected mice.⁴⁷ Some mechanisms of evasion could be intrinsic to the biology of the parasite. For example, the ability of T. cruzi to induce capping and shedding of antigen-antibody complexes from its surface,⁴⁸ must be relevant to avoid FcγR engagement and peroxide formation upon invasion of caMφ. In addition, some studies have suggested that macrophages infected with T. cruzi are defective in antigen presentation to CD4⁺ T cells. In one case, a deficit in protein catabolism and processing was reported.⁴⁹ Another study did not find any defects in antigen processing or class II MHC expression, although physical interactions between macrophages and T cells were reduced.⁵⁰ A deficit in antigen presentation could reduce IFN-γ secretion by interacting Th1 T cells, and failure to generate a caMφ phenotype. However, it is unclear whether these alterations occur in vivo.

Molecules produced by T. cruzi transduce signals to cells of the host immune system.⁵¹ Some of these molecules alert the organism of the presence of the parasite. Glycosylphospha-tidylinositol (GPI)-anchored mucins from T. cruzi activate cytokine release in caMφ by interacting with TLR2.⁵² A T. cruzi released protein related to oxidoreductase family, called Tc52, also activates macrophages and induces dendritic cell maturation via TLR2.⁵³ On the other hand, other parasite molecules appear to have an immunoregulatory role that helps parasite evasion. AgC10 is a GPI-anchored mucin that inhibits macrophage secretion of TNF by inhibiting the activity of p38 MAPK.⁵⁴ The cysteine proteinase cruzipain induces aaMφ differentiation through TGF-β and IL-10 release.⁵⁵ Glycoinositolphospholipid (GIPL) from T. cruzi exerts immunosuppressive effects and counteracts macrophage activation through its ceramide domain.^56,57 Interestingly, pretreatment of macrophages with GPI-anchored T. cruzi mucins reduces subsequent cytokine responses to whole T. cruzi parasites. Furthermore, TLR2-deficient mice produce enhanced levels of cytokines following challenge with live parasites.⁵⁸ These results suggest that GPI-anchored mucins, which bind to TLR2,⁵² might have an immunoregulatory role by reducing proinflammatory reactions to the parasite. The immunoregulatory role of GPI-anchored mucins could be related to another phenomenon, known as LPS desensitization, which involves downregulation of TLR4 signaling.⁵⁹ A recent study suggested that previous LPS desensitization also promotes increased replication of T. cruzi in peritoneal macrophages,⁶⁰ suggesting that interference with TLR signaling compromises macrophage defenses against the parasite. Parasite molecules that subvert immune responses constitute an important obstacle for the development of effective vaccines. Table 1 lists T. cruzi molecules and mechanisms implicated in evasion of macrophage defenses.

Table 1

Parasite molecules and immune mechanisms involved in evasion of macrophage defenses by T. cruzi.

Mechanisms of evasion that modify macrophage activation have been investigated by immunological approaches. The antiinflammatory cytokines IL-10 and TGF-β could be important for evading caMφ. Both IL-10 and TGF-β have deleterious effects that exacerbate infection in vivo.^61,62 In addition, both IL-10 and TGF-β inhibit NO-dependent trypanocidal activity of macrophages primed by IFN-γ.^15,62 TGF-β has potent deleterious effects in vivo, and increases parasitemia, even in animals that have received IFN-γ in vivo.⁶² Both IL-10 and TGFβ are secreted by aaMφ,^6,7 and can induce the differentiation of additional aaMφ.^8,9,12 Therefore, it is likely that T. cruzi evasion relies on generation of aaMφ, which in turn could function as permissive host cells for the parasite.

One mechanism for induction of aaMφ is through secretion of IL-4 and IL-10 by Th2 T cells.⁸ In this regard, Th2-biased mice show exacerbated parasite loads and tissue inflammation, compared to control mice.⁶³ Furthermore, even a residual Th2 response is necessary for both parasite persistence and tissue inflammation in T. cruzi infection.⁶³ Humoral immunity dependent on Th2 T cells is necessary for protection against infection.⁶⁴ Therefore, generation of aaMφ could be an unavoidable by-product of the host humoral immune response. A mixed Th1/Th2 response arises during T. cruzi infection,⁶³ but it is not clear how aaMφ and Th2 T cells are generated. Certain molecules produced by the parasite could play an important role. Cruzipain, a cysteine proteinase produced by T. cruzi, induces a Th2 response in BALB mice.⁶⁵ More importantly, cruzipain induces an alternatively activated phenotype in macrophages even in the absence of T cells, due to increased secretion of TGF-β and IL-10 by macrophages.⁵⁵ Once differentiated, these aaMφ supported increased intracellular replication of T. cruzi,⁵⁵ indicating that aaMφ are permissive hosts for the parasite. Cruzipain increases arginase activity and the subsequent T. cruzi growth by activation of tyrosine kinase, protein kinase A (PKA), and p38 mitogen activated protein kinase (MAPK) activities in macrophages.⁶⁶

A second mechanism for induction of aaMφ is through secretion of the antiinflammatory cytokine TGF-β.^9,12 Secretion of TGFβ has been associated with resolution of inflammation. In fact, induction of aaMφ is regarded as an intrinsic immunoregulatory response of the host, that counteracts potentially harmful Th1 immune responses.⁷ Acute infection with T. cruzi induces an early and potent Th1 response, which is drastically reduced once parasitemia is resolved.⁶⁷ Usually, downregulation of Th1 responses results from apoptosis of effector Th1 T cells mediated by Fas/Fas ligand (FasL) interactions.⁶⁸ Apoptosis of CD4⁺ T cells was described during the acute phase of T. cruzi infection,⁶⁹ and Fas/FasL interactions are involved in death of T cells from infected hosts.⁷⁰ Furthermore, removal of apoptotic cells by macrophages is antiinflammatory, leading to arrest of TNF-α production, and to TGF-β secretion.⁴¹ Therefore, it is possible that phagocytic burial of apoptotic lymphocytes induces aaMφ differentiation, and helps survival and replication of parasites inside TGF-β producing macrophages. To test this hypothesis, macrophages infected with T. cruzi were cocultured with CD4⁺ T cells from infected mice. When T cells were killed by agents that increase FasL expression, or by an agonist anti-Fas antibody, parasite replication inside macrophages was exacerbated.⁷¹ This deleterious effect required physical interaction between CD4⁺ T cells and infected macrophages,⁷¹ suggesting that phagocytosis of dead lymphocytes drives T. cruzi replication inside macrophages. In fact, apoptotic but not necrotic lymphocytes exacerbated T. cruzi replication in macrophages.⁷² The integrin α_Vβ₃ (Vitronectin Receptor) plays a critical role in phagocytosis of apoptotic cells.⁷³ In agreement, both binding of apoptotic lymphocytes, and increased T. cruzi replication could be blocked by an antagonist anti-α_V Fab fragment, and parasite growth could be exacerbated by intact anti-α_V antibodies in the absence of apoptotic cells.⁷² Parasite replication driven by apoptotic cells or by α_Vβ₃ engagement were dependent on PGE₂ and TGF-β production.⁷² Furthermore, PGE₂ and TGF-β upregulated ODC activity and polyamine production. Increased parasite growth relied on putrescine production.⁷² Induction of protracted ODC activity, an enzyme placed downstream to arginase, strongly suggests that apoptotic cell ingestion triggers aaMφ differentiation, and that this was accomplished through delayed effects of TGF-β.⁷² Furthermore, addition of apoptotic cells reverted the trypanocidal effect of Mφ treated with LPS plus IFN-γ, resulting in increased parasite replication.⁷² Therefore, interaction with apoptotic cells is an efficient way to prevent caMφ differentiation. In agreement, injection of apoptotic cells increased and accelerated parasitemia in infected mice.⁷² During the acute phase of infection, the parasite induces intense polyclonal lymphocyte activation that resolves by lymphocyte apoptosis. T. cruzi exploits apoptotic cell clearance and the associated shift in macrophage L-arginine metabolism to replicate inside permissive macrophages.⁷⁴Figure 1 shows the opposing outcomes of T. cruzi replication in macrophages, during infection in the absence or in the presence of apoptotic cells.

Figure 1

Clearance of apoptotic cells shifts arginine metabolism and drives parasite growth in macrophages. Left) In the absence of apoptotic cells, infected macrophages interact with parasite-specific Th1 T cells, leading to IFN-γ production, and induction (more...)

Prospects for the Future

The biochemical pathway that links apoptotic cell removal to intracellular parasite replication provides new targets for therapeutic intervention. For example, treatment of infected mice with blockers of cyclooxygenase,⁷² or with zVAD-fmk, a pan-caspase inhibitor that functions as a general blocker of apoptosis (Lopes MF and DosReis GA, unpublished results), markedly reduces parasitemia in infected mice. In addition, polyamine production is another potential target for chemotherapic intervention against protozoal parasites.⁷⁵ T. cruzi is unable to synthesize putrescine and is dependent on uptake of exogenous polyamines by high affinity transporters.⁷⁶ Polyamines are essential for T. cruzi survival and division. Polyamine analogs block T. cruzi intracellular replication in vitro.⁷⁷ Furthermore, T. cruzi conjugates glutathione with the polyamine spermidine to synthesize trypanothione, a critical antioxidant molecule and a potential target for chemotherapy.⁷⁶ Finally, clearance of apoptotic cells could be involved in tissue fibrosis in situations of exagerated or sustained apoptosis. It has been reported that TGF-β shifts L-arginine metabolism, leading to both polyamine and collagen synthesis.⁷⁸ Together, the results indicate that Th2 cytokines and the clearance of apoptotic cells induce aaMφ, which serve as safe host cells for parasite replication and spread. Since aaMφ upregulate the Arginase/ODC/Polyamine axis, this biochemical cascade is a promising target for therapies aimed at reducing parasite replication, and fibrogenesis secondary to inflammation.

Acknowledgements

Authors' cited work was financed by Brazilian National Research Council (CNPq), Rio de Janeiro State Science Foundation (FAPERJ), and Howard Hughes Medical Institute (HHMI). FLRG is a post-doctoral fellow from FAPERJ. MFL and GADR are investigators from CNPq. GADR is a Howard Hughes International Research Scholar.

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