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Immune Response 17/10/2022

Patients 

The immune response of patients with Chagas’ disease

Cristiane A.S. Menezes

Laboratory of Biology of Cellular Interactions, Department of Morphology, ICB, Federal University of Minas Gerais

Email: cristianemenezes@hotmail.com

Mauro M. Teixeira

Laboratory of Immunopharmacology, Department of Biochemistry-Immunology, ICB, Federal University of Minas Gerais

Email: mmtex@icb.ufmg.br

Walderez O. Dutra

Laboratory of Biology of Cellular Interactions, Department of Morphology, ICB, Federal University of Minas Gerais

Email: waldutra@pq.cnpq.br

Among the many intriguing aspects related to Chagas’ disease, its differential clinical evolution in chronically infected individuals certainly plays an important role. While the vast majority of chronic carriers of Chagas’ disease do not present symptoms or clinical signs of the disease, being classified as “indeterminate”, approximately 40% of chronic patients develop clinical symptoms and laboratory alterations related to the disease. The “digestive” and “cardiac” clinical forms are characterized by tissue damage of varying intensity in the myenteric neuronal network and in cardiac tissues, respectively, and are associated with disease morbidity and mortality. Factors related to the parasite, such as the specific tissue tropism of different isolates of Trypanosoma cruzi, can decisively influence the clinical course of Chagas’ disease. However, the fact that similar parasite isolates can be associated with different clinical forms of the disease points to the importance of other factors, especially the immune response of patients, in its pathogenesis. Undoubtedly, understanding the mechanisms related to the establishment of different clinical forms of Chagas’ disease is a crucial point to guide future prophylactic or therapeutic interventions.

The first studies related to the immune response of patients with Chagas’ disease focused on the humoral response. The detection of antibodies reactive to the parasite was and is an important tool for the diagnosis of infection in humans and, given this association, research has been carried out to better characterize the humoral response of those with Chagas’ disease. Antibodies reactive to galactose epitopes and which can mediate the lysis of trypomastigote forms were found in the serum of chronic patients. These antibodies were named “lytic antibodies” (LA). Recent studies have shown that LA are found at higher levels in the serum of patients with the indeterminate form than the cardiac form of the disease, suggesting a protective role for these antibodies. Studies referring to other classes of antibodies, on the other hand, suggest their participation in the pathogenesis of the disease, since antibodies reactive against different structures of the host are found in patients with chronic Chagas’ disease. Anti-T. cruzi capable of stimulating the proliferation of T and B lymphocytes in patients with Chagas’ disease. These antibodies preferentially stimulate CD5+ B cells, a cell subpopulation related to autoimmune processes. It is possible that antibodies play an important role in the clinical course of Chagas’ disease. Perhaps the fact that the tissue destruction observed in patients with chronic Chagas’ disease is associated with the presence of an inflammatory infiltrate has contributed to the fact that most immunological studies in patients with Chagas’ disease focus on the cellular response. Despite the clear importance of the cellular response in the immunopathological mechanisms in Chagas’ disease, studies that allow a better understanding of the humoral response are essential. After all, considering the complexity of the parasite-host interactions, it is unlikely that only one arm of the immune system is associated with the evolution of the disease.

Pioneering studies on the cellular response of patients with Chagas’ disease showed that peripheral blood mononuclear cells (PBMC) from patients with the indeterminate or cardiac forms are able to proliferate when exposed, in vitro, to parasite antigens and to host components. In addition to proliferating in response to these stimuli, PBMC from patients with Chagas’ disease, especially CD4+ T-cells and monocytes, produce a large amount of inflammatory and anti-inflammatory cytokines. These cells are believed to be fundamental in orchestrating the immune response in patients with Chagas’ disease, influencing the clinical course of the disease. Therefore, many studies have been carried out with the aim of characterizing, phenotypic and functionally, different cell populations and thus understanding their role in the establishment of protective or pathogenic responses against infection with Trypanosoma cruzi.

Most studies concerning the immune response in the acute phase of Chagas’ disease were performed using experimental models. Although a lot of important information has emerged from these studies, caution should be exercised in transferring interpretations to human disease. Studies evaluating the immune response of patients with Chagas’ disease in the acute phase suggest that this phase has a “T-lymphocyte-independent nature”, characterized by the expansion of B cells and activation of monocytes. In agreement with this hypothesis, it is known that the acute phase is characterized by a low proliferative response of lymphocytes, related to the low expression of the receptor for IL-2. Progression to the chronic phase is accompanied by an increase in cellular response. In the chronic phase, there is an increase in the frequency of activated circulating CD4+ and CD8+ T-cells, with high expression of HLA-DR and low expression of CD28. The high frequency of activated CD4+CD28- T-cells in the peripheral blood of patients with Chagas’ disease is associated with the expression of TNF-α and IL-10 in cardiac and indeterminate patients, respectively, suggesting distinct functional roles for these cells. Activated CD8+ T-cells that express granzyme A and TNF-α are predominant in the inflammatory infiltrate associated with cardiac lesions in Chagas’ disease. Local production of cytokines such as IL-15 and IL-7 is believed to contribute to the survival of CD8+ T-cells in cardiac tissue. An interesting finding is that cells with a phenotype consistent with that of regulatory populations, CD4+CD25high and NKT (CD3+CD16-CD56+), were found in the peripheral blood of indeterminate individuals, suggesting that the lack of regulatory populations in symptomatic individuals may lead to exacerbation of cytotoxic activities, culminating in tissue damage.

Functional analysis of T-lymphocytes from patients with Chagas’ disease revealed that these cells express large amounts of the cytokines IL-5, IL-10, IL-13 and IFNγ in relation to uninfected individuals, suggesting the simultaneous existence of anti- and pro-cellular reactivity. -inflammatory in the face of the constant presence of antigenic stimulus, both from the parasite and from the host. Bahia-Oliveira et al. found that treated cured patients with Chagas’ disease had a higher production of IFNγ than those not cured, suggesting a protective role for this cytokine in the healing process associated with chemical treatment. Corroborating this hypothesis, it was recently demonstrated that the treatment of indeterminate patients, at the beginning of the chronic phase, leads to the production of IFNγ. Other authors also suggest a role associated with protection for this cytokine. Gomes et al., found a high production of IFNγ in 83% of cardiac patients and in 59% of indeterminate patients. Furthermore, these authors showed a clear association between IFNγ expression and the occurrence of severe heart disease, suggesting, in the chronic phase, a relationship between IFNγ production and disease morbidity. In this sense, it has already been shown that T-cell clones derived from the inflammatory infiltrate of patients with Chagas’ disease with heart disease are excellent producers of IFNγ. Recent studies have shown a positive correlation between the expression of TNF-α of the chemokine CCL1 and the chemokine receptors CCR5 and CXCR4 with cardiac dysfunction in patients with Chagas’ disease, suggesting an association between TNF-α and pathogenesis. An interesting finding is that, although it is correlated with the establishment of a modulatory profile in patients with the indeterminate form of Chagas’ disease, the anti-inflammatory cytokine IL-10 is also produced by PBMC in patients with heart disease, suggesting that, more than production of a given cytokine by itself, the balance between inflammatory and anti-inflammatory cytokines produced throughout the disease will be critical in determining the course of infection. Thus, factors that determine the production of these cytokines, such as the antigenic stimuli associated with the infection and the occurrence of gene polymorphisms, play an essential role in chagasic immunopathology.

Advances related to the identification of parasite (and host) molecules responsible for inducing immune reactivity in patients with Chagas’ disease have been achieved in recent years. While parasite-derived mucins appear to be essential for triggering the innate response, lymphocyte stimulation occurs in response to a variety of antigens. However, populations of T-cells expressing Vα and Vβ restricted regions in their TCR have already been identified in the peripheral blood and cardiac inflammatory infiltrate of patients with Chagas’ disease, suggesting the existence of a dominant peptide that induces the T-cell response. from the sequencing of the genome of the parasite (and also human), will allow the identification of antigens important in the immune response during the chagasic infection.Based on the studies cited, we can see the complexity of the factors involved in the evolution of Chagas’ disease. Figure 1represents a combination of data from the literature regarding the immune response of patients with Chagas’ disease with the indeterminate and cardiac forms. Although much knowledge has been acquired in relation to the immune response of patients with Chagas’ disease, important aspects have not been clarified yet. The answer to questions such as: (i) which antigens induce pathogenic or protective responses?; (ii) is it possible to prevent the development of severe forms using immunological intervention?; and (iii) can the susceptibility to the development of severe clinical forms be genetically determined? They are important and could direct the search for new preventive and curative therapies, benefiting the infected population or at risk of infection. Transcending to Chagas’ disease, knowledge about the biology of the immune system, obtained from studies in patients with Chagas’ disease, will certainly help in understanding the immunopathology of other infections.

Figure 1

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Cytokines 

Cytokines in Chagas’ disease: a personal perspective on three decades of research

Ises de Almeida Abrahamsohn

Department of Immunology at the Institute of Biomedical Sciences at the University of São Paulo

Email: iabraham@usp.br

Introduction 

The organizers of the International Symposium of the Centenary of the Discovery of Chagas’ disease invited me to write a review on cytokines in Chagas’ disease to be posted on the Oswaldo Cruz Foundation website on the occasion of the Symposium to be held from July 8th to 10th, 2009. The subject is vast and encompasses research published over nearly 30 years. A classic review trying to cite and discuss all the works from this period would be very extensive and possibly of little use to researchers who do not work directly with the subject. For the same reason, I decided not to include the literature on chemokines in Chagas’ Disease. I chose to present my personal view on the development of research on cytokines in Chagas’ disease and Trypanosoma cruzi infection in the last three decades. In the bibliography, representative articles were selected, organized in blocks correlated to the subjects covered in the text. Some articles were included for their historical and/or innovative relevance.

Why study cytokines in Chagas’ disease?

One of the objectives that permeated most of the basic works of the 1980s and 1990s was to identify which cytokines participate in the control of parasitism by the vertebrate host and which cytokines have the opposite effect, that is, they favor greater tissue parasitism. Understanding the role of different cytokines in the protective immune response and in the resistance or susceptibility of the vertebrate host would make it possible to experimentally test the effects of treatment with certain cytokines (or with the respective neutralizing antibodies). The possibility of modifying the course of the experimental infection and increasing resistance was sought. At that time, the future use of cytokines in the treatment of various infectious diseases was predicted. In addition, knowledge of essential cytokines for parasitism control is of paramount importance to develop vaccination protocols against T. cruzi infection.

The second reason to study cytokines in Chagas’ Disease is to understand their action on the various regulatory mechanisms of the immune response observed during infection such as immunosuppression, lymphocyte death and the regulation of the inflammatory response.

The third major focus of research on cytokines in Chagas’ Disease is to understand how these molecules participate in pathogenesis and pathology.

Cytokines and parasitism control by adaptive and innate immune responses

Until the mid-1980s, few cytokines produced in the specific or adaptive immune response were known: IL-2 (called TCGF-T-cell growth factor), the B cell growth factor (BCGF or TCGF 2) that would later be characterized with IL-4 and gamma interferon (also called immune interferon or type II). Between 1985 and 1987, the cytokines IL-4, IL-3 and IL-5 were successively characterized. In 1986, Robert Coffman and Tim Mosmann proposed the existence of two populations of functionally distinct auxiliary T lymphocytes due to the cytokines produced: Th1 lymphocytes producing IFNγ and IL-2 and Th2 lymphocytes producing IL-4, IL-5.

Since the 1970s, extreme variations in the susceptibility of mice of isogenic strains to infection by laboratory strains of T. cruziwere already known. Therefore, since the publication of the Th1-Th2 hypothesis, the role of Th1 and Th2 populations and which cytokines would be associated with resistance and susceptibility has been extensively investigated in infections and also in experimental T. cruzi infection. Interferon-gamma (IFNγ) is essential for the control of parasitism by T. cruzi, as demonstrated by several authors who treated infected animals or macrophage cultures with the cytokine itself or with neutralizing antibodies or even used knock-out animals, devoid of the gene of IFNγ. In addition to IFNγ, also TNF-α (which can also be produced by Th1) participates in infection control. Both IFNγ and TNF-α are potent activators of the microbicidal functions of macrophages, among which are the production of intermediate oxygen (ROI) and nitrogen (RNI) radicals and, therefore, the neutralization of these cytokines in vivo or in vitro increases, respectively, parasitism in the animal or the number of intracellular T. cruzi. Interferon-gamma is the main cytokine responsible for controlling parasitism in the acute phase and also for keeping it at very low levels in the chronic phase.            Differently from what occurs in the infection of mice infected by Leishmania major, it was not possible to show, in the infection of mice by T. cruzi, a dominance of the Th2 type response in BALB/c (susceptible) in contrast to the protective Th1 response of C57BL mice /6 (resistant). Previous induction of a polarized Th2 response aggravates T. cruzi infection and induces the death of resistant animals. However, T. cruzi infection normally done in the laboratory results in intense activation of the Th1 response in both mouse strains. In fact, levels of IFN gamma production by splenocyte cultures are not very different in the susceptible (BALB/c) and resistant (C57BL/6) strains, and IL-4 levels are very low in both. Treatment with neutralizing anti-IL-4 antibodies did not alter the levels of parasites in the blood or the production of IFNγ, suggesting that the IL-4 produced endogenously in the infection is not, in isolation, an important determinant for the susceptibility of mice to T. cruzi.

The discovery in 1989 of IL-10 as a cytokine produced by Th2, with an inhibitory action on the Th1 response, reinforced the idea of reciprocal regulation between Th1 and Th2 populations. Evidence has progressively accumulated that IL-10 is produced by several cells other than Th2 and that it downregulates not only the secretion of Th1 cytokines, but also inhibits and counteracts the activating actions of IFNγ and TNF-α, deactivating the transcription and translation of various genes in macrophages. Furthermore, IL-10 acts by negatively controlling the immune response in general and especially the inflammatory response and tissue damage. The levels of IL-10 produced by mice susceptible and resistant to T. cruzi are not very different, although there may be variations depending on the strains of T. cruzi or mouse studied. However, neutralization of IL-10 by monoclonal Ab increases the production of IFNγ and mice lacking IL-10 (IL-10 KO) have less parasitism, but die earlier with exacerbated inflammation in several organs. In addition to IL-10, splenocytes from T. cruzi-infected mice produce TGF-β which also inhibits macrophage activation by IFNγ.

In fact, the perception has now been consolidated that IL-10, TGF-β and regulatory T-cells (which can produce IL-10 and/or TGF-β) are essential to control the hyperactivation of the immune system and the degree of cell injury. and pathology in various infections.

From the discovery and characterization, in 1991, by Giorgio Trinchieri, of the cytokine IL-12 as an IFNγ-inducing cytokine, produced by dendritic cells and macrophages stimulated by bacterial products, the importance of the innate immune response as essential for the activation of the adaptive immune response and for the organism’s initial resistance to infections. In the innate response, we have IL-12 and TNF-α, both produced by macrophages or by dendritic cells stimulated by T. cruzimolecules that interact with Toll-like receptors (or TLRs) and with other receptors that recognize molecular patterns present in pathogens (PAMPs). The two cytokines, TNF-α and IL-12, are essential for the initial control by the innate response of T. cruziparasitism, along with IFNγ produced by Natural Killer (NK) cells activated by IL-12 and IL-15. However, the susceptibility of mouse strains to T. cruzi infection is not associated with lower IL-12 production.

In addition to the aforementioned cytokines, type I interferons (alpha and beta), long known as antiviral interferons, are classic cytokines of the innate immune response and participate in the initial resistance to infections. Type I Interferon genes, especially IFNbeta, are among the first to be activated after T. cruzi cell infection, and many cell types can synthesize these cytokines. In the 1980s, some studies were carried out on the activity of IFNI in T. cruzi infection. Recent studies have shown that the production of IFNI by macrophages is much higher in the infection-resistant than in the susceptible murine strain, resulting in a lower initial parasite load due to the high production of nitric oxide, which is stimulated by IFNI and also by TNF-α produced by the macrophages soon after their infection. Thus, the initial control of the level of cellular parasitism by the innate response facilitates the general control of the parasitism by the subsequent adaptive response. In addition to IFNI and TNF-α, it is possible that the action of other cytokines such as GM-CSF and MIF are added to those described.

Interleukin 18, another cytokine of the innate response, plays a secondary role to that of IL-12 or even null (depending on the strain of mice studied) in stimulating the production of IFNγ in T. cruzi infection. Recently it was shown that mice lacking the gene for the pro-inflammatory cytokine MIF (macrophage inhibitory factor, a cytokine known for a long time) produce less IL-12 and TNF-α and have more severe infections.

Cytokines in immunosuppression and regulation 

In acute infection by T. cruzi and also in other systemic parasites, one of the phenomena observed since the 1970s is the immunosuppression of the cellular immune response both to the parasite’s own antigens and to polyclonal stimulators of the immune response. I consider immunosuppression one of the regulatory mechanisms to avoid excessive activation of the immune system in the presence of a huge amount of parasitic antigens, as happens in the acute phase. As the infection is controlled by the immune system, the intense immunosuppression of the acute phase tends to disappear, but even in the chronic phase, both in mice and in humans, it is possible to verify that the T lymphocyte response is still negatively regulated by several mechanisms.

The macrophage activating cytokines IFNγ and TNF-α are paradoxically involved indirectly in the suppression of proliferation and death of T lymphocytes and also in the inhibition of the production of several cytokines such as IL-2 and IFNγ itself, phenomena that characterize immunosuppression in acute T. cruzi infection in mice. IFNγ stimulates the production of nitric oxide, which is necessary to eliminate the parasite, but inactivates several proteins and synthesis pathways and ultimately leads to cell death. Another action of IFNγ is to modulate the expression of Fas and Fas-L molecules, also inducers of cell death. The final effector molecules that exert immunosuppression in mice in T. cruzi infection are mainly nitric oxide and secondarily prostaglandins (PG). Experimental inhibition of the synthesis of these molecules associated with the addition of IL-2 restores the proliferation and synthesis of IL-2 and IFNγ by lymphocytes from infected mice.

In patients studied in the chronic phase, it is observed that PG and IL-10 reduce the proliferation of lymphocytes and inhibit the production of several cytokines such as IL-12, IL-2 and IFNγ; the first two are important for the initial activation, proliferation and differentiation of lymphocytes and their supplementation in the culture or the inhibition of IL-10 or PG increases the proliferation of T lymphocytes specific to parasite antigens and the synthesis of cytokines.

A negative regulation mechanism of macrophage activation that favors the survival of the parasite within these cells is the stimulated synthesis of TGF-β after phagocytosis of apoptotic cells in environments rich in inflammatory cells; this mechanism may also be important in the regulation of tissue damage in target organs. Another mechanism in which T. cruzi itself stimulates the synthesis of regulatory cytokines TGF-β and IL-10 has been described in vitro for bone marrow-derived dendritic cells. These DC stimulated by trypomastigote forms of the RA strain develop a suppressor phenotype characterized by the synthesis of TGF-β and IL-10 and reduction of the synthesis of IL-12, correlating with the suppressor phenotype of CD identified in the murine infection by this strain of T cruzi.

Regarding the regulation of the immune response and pathology by regulatory T-cells of endogenous origin (Treg CD4+CD25+), the work carried out in mice has not yet shown a relevant role for these cells.

Another level of downregulation of the immune response could be exerted by fragments of the plasma membrane released by the parasite that form exosome-like vesicles. These have the potential to induce IL-10 and IL-4; in heavily parasitized organs such a mechanism would reduce the microbicidal activity induced by IFNγ and/or TNF-α.

Cytokines and pathology

The third objective in the study of cytokines in T. cruzi infection is to understand their relative participation as determinants of the intensity of inflammation and tissue damage, that is, in pathogenesis and pathology. Variations in parasitism and resistance to T. cruzi infection are observed when different strains of mice are infected by the same strain of the protozoan and mainly in the acute and subacute phases. In the human species, in view of the significant reduction in vector transmission, patients who have an acute symptomatic phase are rarer and, for the time being, there are no means to assess the parasite load in chronic patients.

Great variation in the intensity of pathology is observed among patients in the chronic phase. In Brazil, approximately 70% of the infected have an indeterminate clinical form, without symptoms and with few histopathological lesions, which remain during the life of the individual. In other infected people (30%), the disease manifests itself clinically throughout life and it is observed that the majority have varying degrees of chagasic heart disease; a small percentage develop only megaesophagus and/or megacolon. These data are for Brazil; in other endemic countries or regions, the relative incidences of the clinical forms are different.

Regarding the role of cytokines in pathogenesis and pathology, one of the questions that guides research in patients is to verify if there is a correlation between the expression of certain cytokines and the degree of pathology found in the heart. Mainly, correlations are sought between the synthesis of certain cytokines by mononuclear cells in the blood of patients with Chagas’ Disease and the severity of the clinical form; some studies directly analyzed biopsy or necropsy material. Furthermore, the expression of certain cytokines by peripheral blood leukocytes obtained ex vivo or activated in culture could be used as an indicator of morbidity and progression from the indeterminate form to the symptomatic clinical forms.

Other experimental approaches look for associations between forms or severity of chronic disease with polymorphisms found in cytokine genes in the human species. Once the markers are identified, it would ideally be possible to intervene early with antiparasitic treatment and/or using, for example, humanized anti-cytokine antibodies that could delay the progression of the pathology.

The main cytokines studied were IFNγ, IL-10, IL-4 and TNF-α. The results are far from consensus. In general, it would be expected that patients with higher levels of IL-10 and/or IL-4 (or even TGF-β) or of parasite-specific T lymphocytes that produce these cytokines would have better control of inflammation and tissue destruction. secondary to the anti-parasitic immune response in organs, especially in the heart. In this sense, several works indicate that high levels of IL-10 and moderate levels of IL-12 and/or IFNγ are associated with the indeterminate form; on the other hand, higher levels of IFNγ associated with lower levels of IL-10 and (in some works) of IL-4 are found in cardiac or more severe forms. However, other studies found an inverse correlation between disease severity and the frequency of IFNγ-producing T lymphocytes.

An association was found between more severe forms of heart disease and an allele of the IL-10 gene that results in lower production of this cytokine, suggesting that IL-10 controls the intensity of cardiac injury in humans. TNF-α gene polymorphisms may or may not be associated with symptomatic forms depending on the population studied.

Another approach in a similar direction is the search for the association of clinical conditions with frequencies of regulatory cells producing or not producing cytokines. There are still few studies, but a higher frequency of IL-10-producing Treg cells has already been found in the blood of patients with the indeterminate form of Chagas’ disease compared to patients with cardiac forms.

Approximately twenty-five percent of patients with the cardiac form have higher levels of circulating TGF-β compared to patients with the indeterminate form. Follow-up studies of these patients should elucidate the prognostic value of this finding, considering that this pleiotropic cytokine stimulates fibrosis and has several inhibitory actions on the immune response and on macrophage activation.

Also recently, an attempt has been made to verify the role of the cytokines IL-17, IL-22, IL-23, belonging to the IL-17 synthesis circuit, in the pathology of T. cruzi infection. Another cytokine with negative regulatory action is IL-27 which inhibits the development of Th17 cells, in part due to the induction of IL-10. IL-17 is linked to the induction and maintenance of tissue inflammation in several chronic inflammatory diseases and works together with TNF-α, IL-1, IL-6 and IL-8 and chemokines promoting influx and activating inflammatory cells. Recent data from infected mice suggest that IL-17 is produced concomitantly with the activation of the Th1 response. On the other hand, there is an indication that in the absence of IL-27 signaling, the pathology is much more severe.

The interpretation of association studies between levels of cytokines secreted by blood lymphocytes and degree of pathology requires caution. One problem is that the response of cells found in the blood may reflect neither the composition nor the activation state of the cells that have migrated into the inflamed tissue. Another problem is that patients with heart disease are generally treated with drugs that act on cell receptors also present on lymphocytes and can modify their responses.

In any case, the search for markers, immunological or not, detectable in the blood, that can be used to determine if there is progression of tissue damage remains of great importance. Immune therapies aimed at blocking the progression of tissue damage and treatments to decrease the parasite burden may be selectively indicated for patients who present the markers of progressive disease.

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Apoptosis 

Regulatory role of apoptosis in Chagas’ disease

George A. dos Reis and Marcela F. Lopes

Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro

E-mail: marcelal@biof.ufrj.br

During Trypanosoma cruzi infection, innate and acquired immune responses are carried out by macrophages, T and B lymphocytes and cytokines. These responses control the parasitemia during the acute phase, but the parasite persists for life in host tissues. In experimental models, cytokine-producing CD4+ and CD8+ T-cells are necessary to control parasitemia and to prevent mortality. A balance between type 1 cytokines, which induce microbicidal macrophages, and regulatory type 2 cytokines is necessary to promote immune protection while preventing pathology in cardiac tissue. Type 1 and type 2 responses are coordinated by CD4+ Th1 and Th2 T-cells, respectively. However, in the course of infection, death by apoptosis of lymphocytes occurs and the suppression of T-cell-mediated immunity that follows could contribute to the persistence of the parasite in the host. Apoptosis was detected in the spleen, mesenteric lymph nodes and heart of infected animals, as well as in the heart tissue of patients with chronic Chagas’ disease. Previous studies have suggested a pathogenic role for apoptosis and that blocking it could be a target for therapeutic intervention.

Apoptotic cells and macrophages

Phagocytic removal (epherocytosis) of apoptotic cells by macrophages prevents the release of inflammatory material by dead cells. Furthermore, the ingestion of apoptotic cells induces an anti-inflammatory response due to the production of regulatory cytokines such as IL-10 and TGF-b. We investigated whether phagocytosis of apoptotic cells would affect intracellular replication of T. cruzi in macrophages or not. Macrophages treated with apoptotic cells produced prostaglandins, TGF-b and polyamines, which favored intracellular replication of T. cruzi. In addition, there was inhibition of nitric oxide production, leading to reduced microbicidal activity. Injection of apoptotic cells increased parasitemia in infected animals and treatment with prostaglandin synthesis blockers reduced parasitemia. These results suggest that lymphocyte apoptosis contributes to parasite persistence and that therapies aimed at preventing apoptosis may improve host immune protection against the parasite.

Apoptosis and caspases

Apoptosis is an orderly process of cell death triggered by enzymes known as caspases. It is a response to biochemical injuries or signals provided by cell surface receptors. T and B lymphocytes undergo apoptosis, contributing to reduced immune responses. T-cells undergo two types of apoptotic cell death: passive cell death and activation-induced cell death. Passive cell death is induced by the deprivation of survival factors and is mediated by the mitochondrial cell death pathway under the control of caspase-9. Cell death can also be induced after re-stimulation of T-cells through their antigen receptor (TCR) and is mediated by cell death receptors and caspase-8. Death receptors are encoded by the TNF receptor gene family and their ligands are encoded by the TNF family. The Fas receptor (CD95) and its FasL ligand (CD178) constitute the main, but not the only, pathway of T-cell apoptosis. Trimerization of Fas by FasL leads to the formation of the death-inducing signaling complex, a molecular platform that initiates the process of apoptosis. Active caspase-8 dimers are formed, triggering a cascade of activation of executor caspases that promote cell death. The reduction of the immune response after lymphocyte apoptosis suggests an immunoregulatory role for the Fas pathway and caspase-8. Furthermore, phagocytosis of apoptotic cells by macrophages induces the production of cytokines such as TGF-b, while inhibiting the production of IL-12. These alterations can lead to the down regulation of Th1 responses and the induction of responses mediated by regulatory T-cells (Treg).

Mechanisms of apoptosis in experimental Chagas’ disease 

Both CD4+ and CD8+ T-cells from T. cruzi-infected mice express Fas and FasL and die after activation with TCR agonists. This cell death can be blocked with FasL-specific antibodies. CD8+ T-cells from infected mice also die from factor deprivation and the addition of IL-2 or IL-4 rescues the cells from apoptosis. Active caspase-8 is expressed by T-cells during infection and active caspase-3 is expressed by CD4+ and CD8+ T-cells from infected mice. Caspase blockers such as the peptide zVAD (pancaspase inhibitor) and zIETD (specific for caspase-8) block apoptosis in T-cells obtained from infected animals. Regarding cytokine production, both anti-FasL treatment and caspase-8 inhibition increased the production of type 2 cytokines (IL-4 and IL-10) produced by CD4+ T-cells, and IFN -γ by CD8+ T-cells. These results suggest (i) that CD4+ and CD8+ T-cells undergo apoptosis mediated by Fas/FasL, caspase-8 and caspase-3, and (ii) that inhibition of apoptosis is able to modulate the immune response in T. cruzi infection.

Inhibition of apoptosis during T. cruzi infection

Two strategies were used to block apoptosis in experimental Chagas’ disease: Treatment with anti-FasL or with zVAD, using the diluent or ZFA peptide as controls. Injection of zVAD during the acute phase of infection reduced lymphocyte apoptosis and parasitemia. More lymphocytes were recovered from lymphoid organs and the peritoneal cavity, including activated T-cells. Peritoneal macrophages were more activated, producing more cytokines and NO, but containing less amastigote forms. The immune response was mainly type 1, with an increase in the number of CD8+ T-cells, but without changing the intensity of cellular infiltrates in the cardiac tissue. Interestingly, the treatment of mice with benznidazole, a drug used in the therapy of Chagas’ disease, also induces the accumulation of CD8+ T-cells, due to the inhibition of apoptosis.

Similarly, after treatment with anti-FasL, the number of CD8+ T-cells increased and parasitemia was reduced. After parasitemia was controlled, there was also an increase in the production of type-2 cytokines. These results suggest that anti-FasL antibodies enhance all CD4+ and CD8+ T-cell-mediated responses (Figure 1). Ag-specific CD8 T-cells that express high levels of Fas also express a pro-apoptotic profile correlated with defective immune responses in T. cruzi infection. Furthermore, a viral vaccine expressing a parasite antigen prevented the development of pro-apoptotic CD8 T-cells and induced effective immunity by CD8 T-cells expressing cytokines and cytotoxic activity.

New approaches allowed us to show that CD8 T-cell apoptosis, followed by epherocytosis, alter the functional profile of macrophages. Treatment of CD8 T lymphocytes with anti-FasL promoted anti-T. cruzi immunity, inducing protective M1 macrophages and reducing the M2 response. Therefore, apoptosis and epherocytosis are potential targets to improve the immune response of lymphocytes and macrophages. Therefore, additional studies may reveal new ways to modulate apoptosis and the parasite-specific immune response in the course of Chagas’ disease.

Figure 1. Treatment with anti-FasL improves the immune response against T. cruzi. Mice were treated with anti-FasL during the acute phase of infection to block interactions between the Fas receptor and its ligand – FasL – on the cell membrane (a) and in the soluble form (b). The anti-apoptotic effects of anti-FasL are highlighted in red. (A) Early local immune response. Anti-FasL inhibits apoptosis of CD8+ T-cells, which proliferate and produce IFN-g. IFN-g activates peritoneal macrophages (Mf), which produce NO and kill the parasite. (B) Late systemic immune response. T (CD4+ and CD8+) and B lymphocytes of the spleen express Fas and FasL. Anti-FasL blocks apoptosis of activated B and T-cells, with increased production of Th1 (IFN-g) and Th2-type (IL-4 and IL-10) cytokines, as well as antibodies by B cells.

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Cytotoxicity 

Cytotoxicity in Chagas

Andréa Henriques Pons

Laboratory of Innovations in Therapies, Teaching and Bioproducts, Instituto Oswaldo Cruz/Fiocruz

Email: andreah@ioc.fiocruz.br

Luzia Maria de Oliveira Pinto

Laboratory of Biology of Interactions, Instituto Oswaldo Cruz/Fiocruz

Email: lpinto@ioc.fiocruz.br

General aspects of cytotoxicity

Cytotoxicity is an umbrella term that broadly means induced cell death. It is present in all multicellular and unicellular organisms and in mammals cytotoxicity can be caused by the direct lytic activity of a cytotoxic cell (phagocytic activity and death receptors, for example) or by the secretion of soluble lytic molecules (defensins, components of the complement system, TNF, perforin/granzymes, for example).

In T. cruzi infection, the death of certain cell types would be related to different clinical conditions, contributing to the progression of the pathology. In this context, death of cardiomyocytes in cardiomyopathy, of peripheral nervous system ganglion cells characterizing the neuronal form and of smooth muscle fibers and neurons of the myenteric plexus involved in the “megas”-alterations of the digestive tract has been described. In addition, lymphocyte death is observed in the thymus and in secondary lymphoid organs such as the spleen and mesenteric lymph node. There are also inflammatory lesions in other organs and tissues, such as kidneys, liver and skeletal muscles.

Cytotoxicity in cardiomyopathy induced by T. cruzi infection

Since the acute phase of T. cruzi infection, inflammatory infiltrates associated with apparently necrotic destruction of cardiac fibers are observed. Although it is a commonly observed phenomenon, it is still unclear which cytotoxic effector cells and molecules are responsible for the destruction of cardiomyocytes. However, there are some hypotheses, such as:

Biological cycle

The lesions could be caused by the continuous invasion and proliferation of parasites in the cytoplasm of cardiac fibers. However, the low level of tissue parasitism and pseudocysts after the acute phase are strong arguments against this hypothesis.

Changes in microcirculation

During the acute and chronic phases of the infection, alterations in the coronary microcirculation are observed, which may be involved in the development of myocytolysis and fibrosis. Several constriction points in arterioles punctually associated with numerous microaneurysms were observed in mice infected in the acute phase. Close to the constriction points, there was prominent dilation of the vessel walls, in addition to apparent angiogenesis. There are also microspasms, microthrombi, areas of microdilation, endothelial cell dysfunction, and platelet thrombus formation. In patients with Chagas’ disease, microvascular dilatation was also observed. These vascular lesions can generate punctual ischemia of cardiac tissue due to insufficient coronary blood flow and promote focal necrotic myocardial lesions, interstitial edema, electrocardiographic changes, and eventually death.

Cardiac denervation

Experimental and natural infection by T. cruzi induces extensive intrinsic denervation in different organs that exhibit pathological changes caused by the disease. In this organ there is destruction of parasympathetic ganglion cells, which could generate an imbalance between the sympathetic nervous system (SNS) and the parasympathetic nervous system (PNS). There may also be a correlation between microvasculature constrictions and increased sympathetic activity. Although the PNS has only a mild effect on coronary vasodilation, it is possible that an SNS exacerbation contributes to microvasculature constriction and possible spasmodic events leading to aneurysms.

Participation of innate immune cells

Granulocytes are usually found preferentially in the initial phase of infiltrate formation and have been involved in both the protection and the development of heart disease. Among the granulocytes, neutrophils constitute the most abundant population and it has been shown that in the presence of parasites these cells become activated and are capable of killing even myoblasts in vitro.

Regarding mast cells, there is no consensus on the participation of these cells in the progression of cardiac fibrosis and their relevance in arrhythmia, since most of the data in the literature is restricted to quantitative analysis and the location of cells in tissue sections.

Participation of cytokines and nitric oxide

Recently, it was proposed that some cytokines would have a cytotoxic effect on cardiomyocytes, for example, IFNg and TNF-α (tumor necrosis factor). These cytokines could induce cardiomyocyte death through secondary mediators, such as nitric oxide (NO), reactive oxygen species, cyclic nucleotides or high intracellular calcium levels. It was further demonstrated that the myocyte itself is capable of producing IFNg and TNF-α. Furthermore, it has also been proposed that NO participates in the death of neurons during denervation of the heart and also in the death of lymphocytes. Thus, NO would have a dual role, acting in the control of intracellular parasitism, but also in the death of immunocompetent cells and, possibly, in the progression of cardiomyopathy.

Antibody dependent cell death (ADCC)

For a long time it was proposed that after infection, the immune system itself would recognize cardiac molecules and induce muscle fiber death (autoimmunity), since parasite antigens were rarely observed in the chronic phase of the disease. However, with the advancement of more sensitive techniques such as the polymerase chain reaction (PCR), antigen detection could also be performed at this stage of the disease, and re-aggravation can still be observed in chronic immunosuppressed patients, for example.

Only on in vitro assays was some cytotoxic activity of activated spleen cells observed against infected muscle fibers when serum from chronically infected animals supposedly with autoantibodies (antibody-dependent cell death (ADCC) was added). Other authors have described that antibodies from the serum of mice or chronic patients would recognize muscarinic receptors in isolated rat and rabbit hearts. In this case, there would be no cell death, but alteration of the autonomic cardiac function by the binding of autoantibodies to cardiac receptors.

Participation of cytotoxic T-cells

Among the various proposals for pathways and cells involved in the death of cardiomyocytes in T. cruzi infection, the participation of cytotoxic T-cells is undoubtedly the one that has received the most attention.

Martin and Tarleton, in a recent literature review, and Tzelepis et al. have presented evidence that the response mediated by CD8 T lymphocytes is crucial to the generation of immunity against T. cruzi. However, these cells could contribute to the establishment of chronic myocarditis, since there is a predominance of these cells in the cardiac tissue of T. cruzi-infected mice and also an increased frequency of CD8 T lymphocytes expressing granzyme A in the heart of patients with chronic Chagas’ disease. Thus, the participation of CD8 T lymphocytes in the immunoregulation and/or pathogenesis of T. cruziinfection is still under discussion.

CD8 T lymphocytes mediate different effector mechanisms in fighting infections, such as through the expression and secretion of cytokines and chemokines and/or the induction of cytolytic activity (CTL, cytotoxic T lymphocytes) (reviewed by Andersen et al. in 2006) . In some experimental and human infection models, the functional heterogeneity of the CD8 T-cell population, the inflammatory T CD8 IFNgpos/Granzymeneg versus the cytotoxic T CD8 IFNgneg/Granzymepos, would influence the immunoprotective response.

In this chapter, we intend to present the most recent literature on the role of CTLs in chagasic infection, briefly discussing the role of these cells in protective immunity, presenting data on their involvement in myocarditis and, finally, raising the hypothesis that effector subpopulations of T-cells CD8 would differentially modulate tissue damage.

Identification of LTC response-inducing T. cruzi epitopes 

In 1997, Wizel et al. identified for the first time epitopes of surface antigens of the trypomastigote form (TSA-1) of T. cruzi that were shown to induce a CTL response, conferring protective immunity after transfer of these cells to infected animals. This protective immunity was confirmed by the author and group in 1998 after immunization of different strains of mice with a plasmid containing DNA expressing TSA-1, followed by challenge with T. cruzi. In addition to TSA-1, peptides derived from surface proteins of the amastigote form, ASP-1 and -2 induced a CTL response in T. cruzi infection of mice, according to a study published by Low et al in 1998. In patients with Chagas’ disease, another study by Wizel in 1998 showed for the first time CTL epitopes from ASP-1 and -2 and TSA-1 restricted to HLA-A 2.1. Currently, in vivo cytotoxicity assays using CFSE (carboxyfluorescein diacetate, succinimidyl ester) and labeling with class I MHC (major histocompatibility complex) tetramers constitute new methods for the functional assessment of the specific CTL response. In this context, in assays performed by Tarleton and Martin in 2005, splenocytes from healthy animals were labeled with high or low concentration of CFSE (CFSEHIGH or CFSELOW). While CFSEHIGH cells were placed in contact with a mixture of peptides derived from T. cruzi trans-sialidase (TS), CFSELOW were not. Then, both cells were transferred to chronically infected animals and after 16 hours, cells from chronic animals were isolated and analyzed by flow cytometry. Data showed more than 90% of CFSEHIGHcells were eliminated, indicating specific cytotoxic activity in the chronic phase of infection. Interestingly, Maurício Rodrigues’ group demonstrated that CD8 T-cells specific to TS (H-2Kd) or ASP-2 (H-2Kb) had detectable cytotoxic and IFNg-inducing activity on the 15th day post-infection, after the parasitemia peak, followed by a still detectable decline in the course of the infection.

Interaction of CD4 and CD8 T-cells in cytotoxic activity

In 2007, studies have addressed the dependence of CD4 T-cells on the efficiency of the CTL response in T. cruzi infection. In this context, Tzelepis et al. observed that the expansion of specific CTLs was dependent on the presence of CD4 T-cells and those restricted to MHC class II recognition. In contrast to previous data, a study by Padilla et al. observed that the cytolytic activity of CTLs from MHC class II-deficient and wild-type animals was similar. According to the authors, the presence of CD4 T-cells would be unnecessary for the primary response, but the immunological memory could not be evaluated, since MHC class II-/- animals are highly susceptible.

Activation of the perforin/granzyme pathway

Target cell death through direct interaction with CTL can be induced by perforin, a highly regulated cytolytic pathway involving specific interaction of CTLs with the antigenic peptide/MHC complex in the target cell. The involvement of perforin in T. cruziinfection was initially studied in 1997 by Bisaggio et al. In this study, the authors, when treating the different forms of T. cruziwith purified perforin, did not observe morphological changes or alteration in the infective capacity of the parasite. However, although the parasite itself did not change with the addition of perforin, the infected macrophages were destroyed with the treatment, suggesting an escape mechanism from cytotoxicity by the parasite. In vivo, the role of perforin has been studied in mice genetically deficient in the protein (PKO) infected under different experimental conditions (different inoculum and strains of T. cruzi), resulting in contradictory conclusions by the authors. Thus, Kumar and Tarleton (1998) when infecting PKO, granzyme B (GzmB) deficient animals and wild animals with the Brazil strain observed similar parasitemia, mortality and inflammatory response in all groups of animals in the acute phase, suggesting that the perforin pathway /granzyme is not required to control the infection. In another study, Nickell and Sharma (2000) demonstrated that infection with high inoculum of the Y strain led to uncontrolled parasitemia and increased mortality, while infection with low inoculum induced transient parasitemia and survival of PKO animals, suggesting anti-inflammatory immunity. T. cruzi dependent and independent of perforin. Still using the Y strain of T. cruzi, Henriques-Pons et al. (2002) infected PKO animals and did not observe any change in parasite control in the acute phase of infection, although the PKO animals showed intense cardiac inflammatory infiltrate with a predominance of CD8 T lymphocytes. and cardiomyocyte necrosis, indicating the involvement of perforin in the immunopathogenesis of the infection. In addition to CD8 T-cells, NK cells constitute another important cytotoxic cell population in the initial phase of T. cruzi infection with contact-dependent anti-parasitic capacity, although apparently via a perforin-independent pathway. Unpublished data by Silverio, Oliveira-Pinto et al. showed an increase in the frequency of perforin+ cells in the cardiac tissue of C57BL/6 animals infected in the acute and chronic phases with the Colombian strain of T. cruzi. In this infection model, PKO animals showed higher parasitemia and cardiac parasitism compared to wild animals in the acute phase. In the chronic phase, parasite control was observed in both groups of animals with 100% survival. Thus, it was suggested the involvement of perforin in the protective immunity against T. cruzi, mainly in the acute phase, while in the chronic phase, other immunological mechanisms would be acting to control the infection.

Activation of the CD95/CD95L pathway

Another important cytotoxic pathway is the CD95/CD95L-mediated one, in which CD95L+ cells kill cells expressing the CD95-L ligand (Fas-L), without antigenic specificity (reviewed by Andersen et al., 2006). The first works on the role of the CD95/CD95-L pathway in T. cruzi infection observed increased expression of the receptor and ligand on CD4 T-cells, CD8 T-cells and B lymphocytes, leading to activation-induced cell death (AICD). This increased lytic activity led to an exacerbation of parasite replication and increased parasitemia in both in vitro and in vivo assays.

In an attempt to understand the importance of cytotoxicity via CD95/CD95-L in modulating anti-T. cruzi, Martins et al. (1999) observed that IFNg actively participated in apoptosis, since it induced the expression of CD95/CD95-L after infection. Complementing the previous data, studies in gld/gld or lpr/lpr animals, deficient in CD95-L and CD95 respectively, demonstrated greater susceptibility to infection, compared to wild animals, probably due to the increase in the production of TH2 profile cytokines and decrease in NO. Recently, the role of the CD95/CD95L pathway in the immunoregulation of chagasic infection was re-evaluated in in vivo blockade experiments through the injection of anti-CD95L in infected animals. In animals treated with anti-CD95L, but not with anti-TNF or anti-TRAIL, blockade of the AICD of CD8 cytokine-producing T lymphocytes with a TH1 profile was initially observed, and later, of CD4 cytokine-producing T lymphocytes with a TH2 profile. The accumulation of these cell subpopulations led to efficient control of the parasite.

Thus, it is likely that different pathways of cytotoxicity contribute distinctly and in a complementary manner to anti-T. cruzi as demonstrated in the work of Muller et al. (2003). In the latter, PKO/Gzm-/- or lpr animals infected with the Tulahuén strain showed that parasite control is dependent on the action of perforin and granzymes, but pathogenesis and resistance to infection were controlled by CD95/CD95-L molecules. In this way, the two cytolytic systems would contribute distinctly and in a complementary way in the course of T. cruzi infection.

Caspases in T. cruzi infection

Among the intracellular cell death effector proteins, caspases (reviewed by Siegel et al., 2006) have been evaluated in the context of T. cruzi infection. Using transgenic animals with inhibition of pro-caspase-8 activation or wild animals treated in vivowith a pan-caspase inhibitor, the group of George dos Reis and Marcela Lopes observed parasite control, induction of a TH1 profile response and, more specifically in the last study, activation of cellular immunity and increased frequency of activated profile and memory T-cells. The authors then propose the use of caspase inhibitors as therapeutic targets in the treatment of Chagas’ disease.

Cytotoxicity in the generation of myocarditis and chagasic cardiomyopathy

Different mechanisms of cytotoxicity and soluble inflammatory mediators, as well as the predominance of certain cell populations, would be regulating the balance between a protective tissue inflammatory response, involved in parasite control, and an inefficient or even harmful one, which would lead to tissue injury during infection by T. cruzi. There are certainly still several points to be studied, such as, for example, the demonstration by Leavey and Tarleton (2003) who observed that the majority of CD8 T lymphocytes infiltrating the heart, with activated cell phenotype and/or memory, had attenuated immunological functionality. (IFNg-producing capacity and cytotoxicity), which could contribute to the lack of control of the parasite and a harmful inflammatory response.

The relevance of cytotoxicity in the regulation of chagasic myocarditis has been recently addressed by two studies. In the first, published by Henriques-Pons et al (de Oliveira et al., 2007), the CD95/CD95-L pathway proved to be extremely relevant in the control of myocarditis, since infected gld/gld animals had reduced inflammatory infiltrate in the heart, predominance of CD4 T-cells and less damage to cardiomyocytes. However, gld/gld animals were more susceptible to infection due to renal failure, but not myocarditis.

Unlike previous studies in PKO animals that analyzed only the acute phase of T. cruzi infection, unpublished data from Silverio, de-Oliveira Pinto et al. (2007) demonstrated similar levels of inflammatory cells in the heart tissue of PKO and wild animals. infected by the Colombian strain in the acute phase, but with a predominance of CD4+, IL-4+ and induced nitric oxide synthase (iNOS+) T-cells associated with intense parasitism in PKO animals. In the chronic phase, an increase in the frequency of perforin+ cells was observed in wild animals, but a decrease in IFNg+ cells in cardiac tissue and an increase in cardiac injury markers (such as CK-MB) in serum, compared to PKO animals.

In conclusion, it is possible that there are several cytotoxic mechanisms acting together, in addition to individual factors such as sex, age, nutritional status, family history of kidney and heart diseases and others. Thus, generating a highly complex pathology with regard to the delicate balance between protection versus disease.

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Immunoregulation 

Regulation of the immune response in Chagas’ disease

Luzia Maria de Oliveira Pinto

Laboratory of Biology of Interactions, Instituto Oswaldo Cruz/Fiocruz

Email: lpinto@ioc.fiocruz.br

Vinícius Cotta de Almeida

Laboratory of Cell Biology, Instituto Oswaldo Cruz/Fiocruz

Email: vca@ioc.fiocruz.br

Balance between efficient immune response and immunopathology 

Recovery from an infection requires the production of an efficient immune response that can eliminate or control the pathogen. However, in the period following infection, the induced immune response could also be directly responsible for the generation of pathologies. Trypanosoma cruzi infection represents an appropriate and interesting model to approach the balance between efficient immune response and immunopathology. Despite the important advances achieved in recent years, the regulation of these processes is not completely understood. In this chapter, we intend to present some of the main cellular mechanisms that allow the majority of infected individuals to control the parasite in a non-sterile way, through an efficient immune response, while a small percentage of patients succumb as a result of dysregulation of the immune response. In addition, we will address some of the potential immune mechanisms that are critical to the evolution of infection.

Involvement of “immuno-effector” components in T. cruzi infection

Classic components of the innate immune system, such as dendritic cells (DC), macrophages and NK cells appear to play a crucial role in anti-T. cruzi immunity. In addition, several surface molecules of the parasite were identified as determinants of the activation of innate defense mechanisms. Among these, glycosylphosphatidylinositol (GPI) anchors should be highlighted. They are GPIs covalently linked to mucin-like glycoproteins (GPI mucins) and the trans-sialidase (TS) enzyme (as reviewed by Gazzinelli and Denkers, 2006). In the innate immune system, pattern recognition receptors (PRRs) expressed on antigen-presenting cells (APC), macrophages and DC recognize the molecular patterns associated with pathogens (PAMPs). Among the various PRRs, Toll-like receptors (TLR) are the most studied. Each member of this family recognizes some essential components present in different microorganisms and, acting together, these receptors can recognize most pathogens.

Several studies have addressed the involvement of TLRs in T. cruzi infection. Campos et al. (2001) observed that macrophages from TLR2-deficient mice (TLR2-/-) do not produce pro-inflammatory cytokines after stimulation with GPI. Similarly, macrophages isolated from TLR6-/- mice were unable to respond to GPI stimulation, suggesting that the TLR2-TLR6 heterodimer complex and the CD14 molecule may be responsible for GPI recognition. In vivo, GPI-containing ceramide triggers the production of chemokines, such as the chemokine ligand CXC-2 (CXCL2), in wild-type mice, but not in animals that express a non-functional TLR4. TC52, another T. cruzi-derived protein, also induces the synthesis of pro-inflammatory cytokines in host cells through TLR2. Furthermore, T. cruzi DNA, possibly through unmethylated CpG DNA motifs, can stimulate macrophage and DC activation in a TLR9-dependent manner.

APC activation via TLRs 2, 4, 6 and 9 triggers phosphorylation of mitogen-activated protein kinase (MAPK) – an inhibitor of nuclear factor κB (IB), resulting in the induction of pro-inflammatory cytokine genes such as TNF, IFNg, IL-12, which are important controllers of parasite replication. It was also demonstrated that CD obtained from infected animals show decreased maturation, migration and MHC class I antigen presentation. These data suggest that such a deregulation would compromise the cellular response, facilitating the persistence of the parasite in the host.

The role of macrophages in the response to an infection is also crucial, since this is one of the main target cells for the parasite and, due to its infection resistance activity, mainly related to the production of pro-inflammatory cytokines (for example, TNF-a) and microbicidal components (for example, nitric oxide – NO).

As an initial primary source of IFNg in acute T. cruzi infection, NK (natural killer) cells play a key role in parasite control. NK cell-derived IFNg potentially increases IL-12 production in macrophages, promoting NK cell activation and parasite-specific Th1 cell differentiation, as well as inhibiting the Th2 cell response. In fact, depletion of NK cells has been shown to result in increased parasitemia and mortality in mice in the acute phase of infection. On the other hand, the administration of IFNa and -b, which are able to stimulate the activity of NK cells, induced resistance to the parasite.

Particularly, it is important to mention the studies on the role of gd T-cells and NKT-cell subsets, since these small subpopulations of T-cells have immunomodulatory roles in infections. Depletion of gd Vg1 T-cells appears to upregulate the Th1 response during T. cruzi infection, as seen from decreased IFNg by CD4+ and CD8+ T-cells, which is associated with reduced cardiac inflammation and increased mortality in infected animals.

The role of NKT-cells, which produce high levels of cytokines after stimulation with glycolipids (which bind to CD1d, MHC class I molecules), has recently been addressed in T. cruzi infection. However, its role remains controversial. One study showed that after infection with the CL strain of T. cruzi, mice deficient in all types of NKT-cells showed moderate anti-parasite response and cardiac inflammation, whereas mice deficient in classic iNKT-cells (cells that carry the invariant Va14Ja18 from TCR) showed a strong inflammatory response and increased mortality. It was later shown that C57BL/6 mice infected with the CL strain of T. cruzi and pretreated with α-galactosyl-ceramide (GalCer-α, a glycolipid that binds to CD1d) were protected against infection in a IFNg-dependent, mediated by iNKT, CD4+ and CD8+ T-cells, and phagocytes, but independent of NK cells, suggesting an important role for classical NKT-cells. On the other hand, a previous report with CD1d-deficient mice (ie, deficient in all NKT-cells) infected with the Y strain of T. cruzi showed that these mice behaved similarly to the control mice.

The adaptive immune response, with the combined action of CD4+ and CD8+ T lymphocytes, as well as B lymphocytes, proved to be critical for resistance against the parasite through cytokine secretion, cytotoxicity or the secretion of specific antibodies. Despite the central role of CD4+ T-cells in immunity and in the production of anti-T. cruzi, its presence as an essential contributor to the generation of an efficient immune response has recently been challenged. In the study by Padilla et al. (2007), both MHC class II and C57BL/6 deficient mice infected with the Brazil strain of T. cruzi showed an increase in the number of CD8 T-cells specific for the TSKB18 and TSKB20 peptides of the trans-sialidase. In addition, MHC class II-deficient mice showed a specific functional response of CTL (cytotoxic T lymphocytes), as observed from IFNg production and cytotoxic activity, even though the cells have a lower migratory capacity in peripheral tissues. In contrast, Tzelepis et al. (2007) reported that mice infected with the Y strain of T. cruzi have the expansion and differentiation of T. cruzi-specific CD8+ cytotoxic T-cells dependent on both parasite replication and CD4 T-cells. + MHC class II restricted.

Immunoprotection mediated by CD8+ T lymphocytes in T. cruzi infection was evidenced, for example, in studies with CD8-deficient mice, which rapidly succumb to infection, and show an increase in parasitism and absence of inflammatory response in tissues. However, the role of CD8+ T-cells in different host compartments during T. cruzi infection remains poorly understood. Currently, new methods for the functional assessment of the specific CTL response have been established, resulting in advances in this area. These methods are in vivo cytotoxicity assays using CFSE (carboxyfluorescein diacetate, succinimidyl ester) labeling, the assessment of IFNg-producing T-cells by ELISPOT (immunospot) or by flow cytometry, as well as the assessment of the frequency of Specific CTL by staining with MHC class I tetramers. The latter allowed the identification, for example, of a high frequency of CD8+ T lymphocytes specific for the immunodominant peptides of T. cruzitrans-sialidase, which can be detected in chronic phase of infected animals and carriers of Chagas’ disease. In addition to frequency, the efficiency of CD8 T lymphocytes in different target tissues is also a subject of study. Leavey and Tarleton (2003) demonstrated that most CD8+ T-cells present in muscle tissue are cells that express cell surface markers of memory and effector function, but have a markedly attenuated effector function compared to their splenic counterparts. The dysfunction of CD8+ T-cells in muscle tissue suggests a mechanism by which T. cruzi may persist in this location and cause inflammatory damage.

In a study with animals chronically infected with the Brazil strain, Martin and Tarleton (2005) observed that splenic CD8 T-cells, after in vitro specific antigenic restimulation, showed a phenotypic profile of fully differentiated memory cells, with homeostatic proliferation. , IFNg production and cytotoxicity. These studies indicate a difference in cellular functionality depending on the tissue where the cells are located.

Reinfection studies have also contributed to the understanding of the effectiveness of anti-T. cruzi immunity. Marinho et al. (2004) observed an increase in the number of IFNg-producing splenocytes, accumulation of macrophages and CD, as well as an increase in the frequency of B, T CD4+ and CD8+ lymphocytes in the spleen of mice infected with the same strain one year after transplantation. primary infection. In addition, the authors observed subpatent parasitemia, indicating the efficiency of the mechanisms of action against T. cruzi; however, chronic lesions were not evaluated in this model.

In a study of patients with Chagas’ disease grouped according to different degrees of cardiac dysfunction, Albareda et al. (2006) observed that individuals with no or mild cardiac involvement were more likely to mount IFNg-producing T-cell memory responses specific to the T. cruzi than subjects with advanced heart disease. In addition, the T. cruzi-specific CD8+ T-cell population was enriched for early differentiated cells (CD27+CD28+) in responders. In contrast, the frequency of CD27+CD28+ CD8+ cells in the total population of CD8 memory T-cells decreases as the disease becomes more severe, while the proportion of fully differentiated CD8+ memory T-cells (CD27-CD28-) increases, which suggests increased presence of cells entering clonal exhaustion. Maintaining a higher frequency of memory T-cells in a secondary response implies more efficient control of the pathogen in reinfection. In this sense, finding a regimen for the generation of efficient memory T-cells may be necessary for future vaccination strategies.

The involvement of CD8+ T lymphocytes in the generation of chronic myocarditis and heart disease has been addressed, since these cells predominate in inflammatory infiltrates present in the cardiac tissue of infected mice with chronic Chagas’ disease. Several reports suggest that the preferential migration and accumulation of these cells in target tissues may be related to the increased expression of cytokines such as IFNg, TNF, IL-6 and IL-4, class I and HLA II molecules, and of adhesion molecules and chemokines induced by IFNg and its receptors (MCP-1, IP-10, MIG and CCR2, CCR5 and CXCR3). Furthermore, Fonseca et al. (2007) reported that the accumulation of CD8+ T-cells may be related to a preferential response to growth factors such as IL-7 and IL-15, given that, in cardiac lesions in chronic patients, these cells express high levels of IL-15 receptor α chain (IL-15R a) and cytokine g chain (CD132).

Interestingly, Cardillo et al. (2007) open a new contribution from B lymphocytes in immunity against T. cruzi. After infecting mice lacking mature B cells with low inoculum of the Tulahuem strain of T. cruzi, they observed a reduction in the frequency of central memory (CD44high/CD62Lhigh) and effector (CD44high/ CD62Llow) in the spleen and in the inflammatory infiltrate of skeletal muscle (CD8+CD45RBlow) of these animals. In addition, these animals had a reduced inflammatory infiltrate and an increase in the number and size of parasite nests, suggesting a poor activation of CD8+ infiltrating lymphocytes and, consequently, a lower resistance to infection in the absence of B lymphocytes.

Recently, Marinho and colleagues (2007) reaffirmed the crucial role of IFNg in anti-T. cruzi immunity. In a study of immunocompetent and immunodeficient mice infected with a low virulence strain, Sylvio X10/4. In contrast, with experimental models using highly virulent strains that result in host death in acute infection, the authors observed a relative contribution of NO and CD4+, CD8+ and CD28+ cells and confirmed the role of IFNg. The authors suggest a compensatory effect of IFNg in immunodeficient animals, as this cytokine induces several immunoprotective mechanisms, including macrophage trypanosomicidal activity (through NO production), MHC class II induction and TLR expression, T-cell polarization CD4+, targeting the isotype response to IgG2a and chemokine production.

The involvement of immunoregulatory cellular components in the progression of T. cruzi infection

A current concept about the chronic course of T. cruzi infection shows an exacerbated deleterious inflammatory response in target organs as a result of a specific immune response to the few parasites in these organs. Among the exacerbated inflammatory components, high levels of IFNg appears to be critical. This postulate seems paradoxical, considering that an effective anti-parasite response is crucial to control the infection and the consequent progression to severe clinical forms of the disease. In fact, Laucela et al. (2004) demonstrated an inverse correlation between the severity of Chagas’ disease and the production of IFNg by CD8+ T-cells in response to T. cruzi peptides or total parasite lysate. These results, together with the finding of increased immune response in patients living in endemic areas, suggest that IFNg production by memory CD8 T-cells is crucial for infection control and progression to severe symptomatic clinical forms of the disease. . However, at this point, it is important to emphasize the critical role for some of the various immunoregulatory components capable of controlling a possible deleterious response following an effector immune response.

Previous studies have revealed the role of the immunoregulatory cytokines IL-10 and TGF-β in the course of T. cruzi infection. The presence of these cytokines was related to an increased susceptibility to infection, possibly related to the inhibition of IFNg-mediated trypanosomicidal activity. In fact, the association between effector and pathological mechanisms in response to the parasite can be observed with studies of a more effective anti-parasite response associated with increased inflammation and mortality in mice deficient in IL-10 or IL-27 signaling. In this context, some studies have compared the participation of both regulatory and effector subtypes and between asymptomatic patients and patients with the severe cardiac form of Chagas’ disease. Gomes et al. (2003) observed a lower frequency of IFNg-producing T-cells in the group of asymptomatic patients. Furthermore, grouping these patients as high and low IFNg producers revealed that the low producers had increased numbers of IL-10-secreting CD14High cells.

The participation of immunoregulatory T-cells with immunosuppressive activity, such as NKT-cells (CD3+CD56+) and regulatory T-cells (natural CD4+ CD25HighFoxP3+), was also addressed. Vitelli-Avelar et al. (2005) demonstrated that asymptomatic patients showed an increase in the frequency of these regulatory cells concomitantly with a high number of effector T-cells and NK cells. Likewise, the lower frequency of these cells in patients with the severe cardiac form of the disease is related to the increase in the number of CD8+ effector T-cells. In addition, asymptomatic patients showed an increased frequency of IL-10+ cells within the CD4+ CD25HighFoxP3+ subset. However, a recent study in mice suggests that CD4+CD25+ cells are not crucial in the immunoregulation of anti-T. cruzi response, since the depletion of CD4+CD25+ cells did not result in any change in the course of acute or chronic infection.

A last aspect to be mentioned is the modulation of the specific immune response after treatment with the anti-T. cruzibenznidazole (Bz). Children with asymptomatic Chagas’ disease treated with Bz showed an increase in the number of CD8+ and NK cells high producing IFNg, along with increasing numbers of CD4+IL-10+ cells and B lymphocytes. These data point to a fine regulation in which anti-parasite effector cells and immunosuppressive cells are concomitantly modulated by Bz treatment.

Conclusions

Here, we summarized some of the data in the literature about regulatory components present in the anti-T. cruzi effector and efficient immune response, and in its deregulation. Overall, the immune mechanisms resulting from the various cellular interactions that play a role in the anti-parasite response, together with the role for the genetic susceptibility of the host, are possible to produce a highly complex pathology that imposes difficulties for the generation of effective vaccines and other immunotherapies. Therefore, in addition to the development of anti-parasite drugs, new therapeutic strategies should include the regulation of cellular immune function and inflammatory components as critical targets in Chagas’ disease.

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