Laboratory of Interactions Biology, Oswaldo Cruz Institute/Fiocruz
Chagas disease is an important parasitic disease caused by the hemoflagellate Trypanosoma cruzi, affecting 16-17 million people in Latin America, as reviewed by Moncayo in 2003. The Chagas’ infection acute phase can be asymptomatic or characterized by patent parasitemia, and may be accompanied by fever, malaise, lymphadenopathy, and inflammation at the site of infection, as first described by Carlos Chagas in 1909. The heart is the most affected organ in the acute phase, and some individuals (3-10%) may present severe and eventually fatal myocarditis. Despite this, in most cases clinical manifestations are not found in the acute phase. Most patients develop the indeterminate form of the disease, showing no clinical signs. However years or decades after infection 10-30% of patients present with one of two main clinical manifestations: (i) heart disease associated with myocarditis and fibrosis, resulting in heart failure, thrombus formation, and cerebrovascular accidents; and (ii) digestive changes such as megacolon and/or megaesophagus, which may be associated with gastro-intestinal disturbances such as regurgitation, malnutrition, and severe constipation, as reviewed by Rossi and Mengel and Higuchi and collaborators.
The immune response establishment that results in an inflammatory process in target tissues during the T. cruzi infection acute phase is essential for the parasitism control and parasite/host relationship balance, which is observed in most Chagas disease carriers. However, in about 30% of patients the inflammation becomes progressive, resulting in cardiac and/or digestive dysfunction. The parasites scarcity in the affected tissues and the apparent lack of correlation between their presence and the inflammatory infiltrate occurrence in these tissues gave rise to some theories such as the parasympathicopriva theory, presented by Köberle in 1958, and that of autoimmunity, recently reviewed by Leon and Engman and Cunha-Neto and collaborators. However, results recent interpretations showing the existence of autoimmune processes in T. cruzi infection have cast doubt on the autoimmune recognition relevance in the chagasic infection chronic manifestations pathogenesis, as reviewed by Kierszenbaum. Also, the involvement of thrombo-embolic phenomena in the chronic Chagas’ disease pathogenesis has been proposed by Rossi and collaborators.
The genetic material detection (by PCR) and antigens (by immunohistochemistry) of T. cruzi in association with inflammatory infiltrates suggests the direct parasite participation in the tissue lesions formation and perpetuation and loss of organ functionality in Chagas disease. There is also more recent evidence that parasite persistence in tissues associated with maladaptive homeostatic mechanisms, such as oxidative/anti-oxidative and pro-inflammatory/anti-inflammatory processes associated with immune response dysregulation are critical for the Chagas’ heart disease formation and progression, as reviewed by Higuchi and collaborators and Lannes-Vieira.
It is now believed that the Chagas disease different clinical manifestations are the consequence of multiple factors linked to T. cruzi, such as (i) strain, (ii) virulence, (iii) antigenicity, (iv) tropism, and (v) inoculum size, and to the host, such as (i) age, (ii)sex hormone-related characteristics, (iii) genetic characteristics, and (iv) previous immune status and that resulting from co-infections. Thus, the molecular mechanisms involved in the pathogenesis of the chronic manifestations in Chagas disease that result in the cardiac form, the digestive form with megacolon and/or megaesophagus formation, as well as the nervous form remain under discussion.
Laboratory of Interactions Biology, Oswaldo Cruz Institute/Fiocruz
Since the American trypanosomiasis discovery by Carlos Chagas, much effort has been expended in trying to understand the chronic Chagas’ disease intriguing pathogenic mechanisms, the leading cause of death and disability in individuals infected with T. cruzi.
Historical aspects of the Chagas disease cardiac form discovery can be found in the section “The cardiac form of Chagas disease – history”. The cardiac form pathology characterization and clinical changes of Chagas disease can be found in the section “The cardiac form of Chagas disease – current reality and functional assessment”.In a recent study Freitas and collaborators demonstrated that etiology is the main prognostic factor for the risk of death in the heart disease case. They observed that Chagas disease presents the highest death risk when compared to heart failure of idiopathic, ischemic or hypertensive origin, conditions that do not present the chronic inflammatory component observed in the chagasic infection. However, the pathophysiological factors that control the cardiac inflammation formation and perpetuation in Chagas disease patients remain to be clarified. Chagas’ infection Acute myocarditis is characterized by (i) the intense parasitism presence, (ii) destruction of parasitized cardiac muscle cells, and (iii) mononuclear inflammatory infiltrate presence, being directly attributed to the parasite and the humoral and/or cellular immune response directed to its antigens. On the other hand, chronic myocarditis of chagasic infection is distinguished by presenting (i) polyfocal inflammatory infiltrate with mononuclear cells, (ii) myocardial fibers destruction in the inflammatory focus, (iii) fibrosis areas, and (iv) parasites rare finding, with no correlation between parasitism and inflammatory reaction, as initially described by Vianna in 1911 and Chagas in 1916 (Figure 1).
Thus, several theories have emerged in an attempt to explain the heart disease pathogenesis associated with Chagas disease. Below are reviewed some historical and current aspects of these attempts to explain the chronic chagasic cardiac dysfunction pathogenesis. It should be considered that even though there are data and arguments supporting these theories, they are not mutually exclusive and the immunopathogenic mechanisms that support them could collectively contribute to the final heart disease picture. Also reviewed are the main players of the cardiac inflammatory process, such as cellular and molecular components, with cytokines, and the pathophysiological processes, such as fibrosis and apoptosis, proposed to participate in the Chagas’ heart disease pathogenesis. Finally, gene polymorphism studies that attempt to associate host characteristics with susceptibility, progression, and risk of death in chronic Chagas’ disease are reviewed.
The autonomic nervous system involvement in Chagas disease was first proposed by Carlos Chagas in 1913. However, the first description of lesions in cardiac autonomic nervous ganglia and fibers was made by Möckenberg, in 1924, using the experimental infection model in dogs. But it was Köberle who carried out pathological studies in Chagas disease patients in the 1950s and 1960s that demonstrated the important involvement existence of the autonomic nervous system in Chagas disease, especially the parasympathetic. These studies and the research current aspects in this area have been reviewed by Rocha and collaborators in the Autonomic Tests section in the chapter Methods of non-invasive functional assessment of Chagas’ disease.
The hypothesis of the autoimmune recognition central role in the pathogenesis of Chagas’ disease that emerged in the 1970s has had a negative impact for more than 20 years on research into new trypanocidal drugs or vaccines for Chagas’ disease. The development of these became considered a waste of time since the autoimmune processes would be triggered by T. cruziantigens early in the infection. In this sense, vaccine components could contribute to the infection worsening they were supposed to prevent, as critically reviewed by Kierszenbaum in 2005. But how did this start and what actually demonstrates and supports the existence of autoreactive processes and autoimmunity in the Chagas‘ infection? What role would autoimmune processes play in the Chagas disease pathogenesis?
Margarinos-Torres in 1929 and Chagas in 1934 proposed that the intense inflammatory lesions associated with the parasites absence in the chagasic infection chronic phase would originate from “immune-allergic mechanisms” activated by products released by the degenerated parasite. Later, Kozma et al. in 1960 proposed that during the infection acute phase intensely parasitized myocardial fibers would rupture, releasing their own intra-cytoplasmic antigens, naturally not accessible to the immune system, which would trigger an autoimmune reactions cascade with the anti-myocardial antibodies production.
Experimental evidence for the autoimmune processes participation in the inflammatory processes genesis found in the myocardium of Chagas disease patients emerged with the cell-mediated immune response demonstration against both parasite antigens and self antigens in animals immunized with T. cruzi antigens. Also, infected patients and rabbits were shown to exhibit lymphotoxicity against parasitized or non-parasitized cardiac fibers by direct recognition and cardiac fibers destruction mediated by activated T cells, as shown by Santos-Buch and Teixeira, or by antibody-dependent cellular cytotoxicity, as shown by Laguens and collaborators. Thus, the authors proposed that autoimmune mechanisms would be established soon after infection and would be perpetuated by continued antigenic stimulation during infection. Despite the impact that these findings caused in studies on the chagasic myocarditis pathogenesis, other authors such as Mortatti and collaborators failed to demonstrate increased autologous cardiac fibers recognition in the anti-T. cruzi antibodies presence or absence. However, the autoimmune mechanisms participation in the chronic chagasic myocarditis genesis found support in studies showing the antibodies presence (of the EVI type), present in Chagas disease patients serum, which recognize endothelial cells and plasma membranes of cardiac and skeletal muscle fibers, as published by Cossio and collaborators. Also supporting this hypothesis is the antibodies presence demonstration in T. cruzi infected patients and experimental animals that recognize (a) neuron/glia by Ribeiro dos Santos, (b) peripheral nerve by Khoury and collaborators, (c) myelin basic protein by Chaves and collaborators and Al-Sabbagh and collaborators, (d) lymphocytes by Ribeiro dos Santos and Buch, (e) thymocytes and components of the thymic microenvironment by Savino and collaborators. However, many of these papers should be considered carefully, because EVI-type antibodies recognize the Gala1-3Gal rodent laminin molecule epitope, an extracellular matrix glycoprotein, shared by an epitope present in a T. cruzi glycoprotein, as demonstrated by Szarfman and collaborators, Gazzinelli and collaborators, and Umezawa and Kanbara. On the other hand, in many papers in which the reactivity of patient serum antibodies against proprietary antigens is supposed to be demonstrated, the assays were performed with antigens and tissues from rats, rabbits, mice, and other animals as the substrate, thus reflecting allo-reactive rather than auto-reactive responses.
Still, trying to explain the autoimmune processes involvement in the Chagas’ myocarditis genesis, Ribeiro dos Santos and Buch proposed that in the first days after infection, antigens from the amastigotes would be released in the vicinity of cardiac fibers or neurons, so that these cells would be sensitized by these antigens. Thus, the onset of humoral and/or cellular immune response against the parasite would determine the cells sensitized destruction by the parasite antigens. On the occasion of the sensitized normal cells destruction there would occur the autoantigens release (or cryptic antigens) and consequently an autoimmune reaction. Such a reaction would depend on a genetic predisposition of the individual or experimental animal and would be responsible for ongoing tissue aggravation or destruction. Experiments have supported this possibility by showing that muscle fibers and neurons are susceptible to in vitro sensitization by antigens released from infected cells or T. cruzi microsomal fractions, these cells being lysed by lymphocytes or granulocytes of the eosinophil and neutrophil types in dependence on antibodies that recognize parasite antigens and complement, as shown by López and collaborators.
On the other hand, Hudson in 1985 proposed that cross-reaction between parasite and cardiac cells antigenic epitopes might be responsible for the autoreactive T lymphocytes activation in Chagas’ infection. The autoreactive clones would be perpetuated after undergoing polyclonal activation during the infection acute phase, and this, according to Petry and Eisen, is the most likely mechanism for the development of autoimmune processes in Chagas disease. Supporting this hypothesis is the cross-reactivity demonstration between ribosomal proteins and parasite antigens, described by Levin and Bonfa and collaborators and of molecular mimicry between a 160 kD antigen associated with the T. cruzi flagellum and a 48 kD protein from the host nervous system, described by Van Voorhis and collaborators.
More recently, an epitope of 6 amino acids from the myosin molecule heavy chain has been identified that shows homology with an immune-dominant antigen from T. cruzi (the B13 protein). Furthermore, Cunha-Neto and collaborators demonstrated that 100 % of sera from chronic Chagas disease patients with heart disease have antibodies that react with cardiac myosin and B13, whereas only 14 % of asymptomatic patients have this cross-reactivity. In addition, the authors showed that CD4+ T cell lines obtained from cardiac fragments of cardiac chronic Chagas disease carriers respond to in vitro proliferative stimulation to both proteins. However, it has not been clarified whether the cross-response is a recognition result of both proteins by a single clone or the several clones existence with different antigenic specificities in T cell lineages. In any case, myosin is an intracellular antigen and the recognition participation of this antigen by antibodies or cells in the heart disease pathogenesis has not been demonstrated, and this self-recognition may be an important physiological mechanism for cellular material removal after parasite-induced lysis.
Further evidence supporting the autoimmune processes existence in the chagasic infection chronic phase arose from the experiments performed by Laguens and collaborators and Said and collaborators who transferred parasite-free T cells from chronically infected animals to normal mice, leading to the inflammatory lesions generation in the heart and central and peripheral nervous tissue, mimicking chronic lesions. Hontobeyrie-Joskowicz and collaborators showed that CD4+ T lymphocytes displayed a delayed hypersensitivity-type immune response against T. cruzi and irrelevant antigens (sheep RBCs, for example), cross-responding to parasite and peripheral nerve antigens. However, later experiments did not confirm such results, suggesting that self-reactive T lymphocytes against cardiac components arise during chronic infection as a tissue damage result, as proposed by Gatass and collaborators.
The most convincing evidence supporting the autoimmunity participation in the Chagas’ myocarditis development was brought by Ribeiro dos Santos and collaborators who showed the rejection of neonate hearts transplanted into the mice chronically infected ear with T. cruzi with kinetics similar to those presented by allografts, this rejection being mediated by CD4+ cells. However, Tarleton and collaborators, using the same model, failed to reproduce Ribeiro dos Santos’ findings and showed that the parasite presence in the heart tissue is “necessary and sufficient” for rejection of the transplanted myocardium to occur. Previous experimental evidence already pointed in this direction, as reviewed below.
At this point, it should be considered that the presence of autoantibodies and self-reactive T cells has been demonstrated in normal individuals at low frequency since the 1950s, these cells frequency being associated with tissue injury processes, such as myocardial infarction. It is believed that these antibody cells presence is associated with a cellular debris removal physiological mechanism, preventing the tissue injury spread, or even a homeostatic mechanism of tumor cell control by apoptosis induction, which would be dysregulated in individuals presenting autoimmune diseases, as reviewed by Schwartz and Kipnis and Toubi and Shoenfeld. How these new insights into autoimmune recognition apply to understanding the Chagas pathogenesis remains to be explored.
In any case, as critically reviewed by Kierszenbaum, the passionate controversy surrounding the hypothesis of autoimmune recognition involvement in the Chagas disease pathogenesis has had the consequence of discouraging the development of new chemotherapy and vaccine against T. cruzi.
B and T lymphocytes polyclonal activation, observed in the chagasic infection acute phase, is also considered to be a triggering pathology factor found in the chagasic infection chronic phase, as proposed by D’Imperio-Lima and collaborators and Minoprio and collaborators. The authors propose that the T cells proliferative activity and the B cells intense polyclonal activation during the experimental T. cruzi infection acute phase leads to the production of IgM- and IgG-class antibodies with reactivity, and even multi-reactivity, against myosin, myoglobulin, keratin, and other proprietary proteins. Thus, such polyclonal expansion could result from the activation of cell clones reactive to a wide variety of parasite antigens or to superantigenic nature molecules from the parasite, as proposed by Minoprio and collaborators and Leite-de Moraes and collaborators. This process could play a role in the loss of tolerance that would precede the autoimmune response in chronic Chagas disease.
Supporting this proposal, using normal human peripheral blood cells for infection by T. cruzi in vitro with the CL strain, Van Voorhis observed activation of lymphocytes, monocytes and cytokine production, suggesting that infection by the parasite triggers in the host a polyclonal response that may be responsible for the pathology observed in the chronic phase. According to this and other authors, such as Coutinho, T. cruzi infection could disturb the immune response regulation during the acute phase, and may generate an imbalance in the autoantibodies idiotypic network and natural self-reactive T cells, contributing to the lesions perpetuation in the chagasic infection chronic phase.
Recently, the research group led by Minoprio showed that proline racemase, an enzyme that participates in the differentiation into T. cruzi infective forms and its penetration into host cells, when secreted activates host B cells in a polyclonal manner and prevents the specific humoral immune response, favoring the parasite evasion and its persistence in the host. Thus, the identification of molecules and the mechanisms understanding that lead to polyclonal activation during T. cruzi infection may contribute to the vaccines development, as well as to the identification of therapeutic targets and the trypanocidal or immune-modulating drugs rational design.
In human Chagas’ infection and in the murine infection model, it is possible that microvascular alterations resulting from thromboembolic processes play a relevant role in the chronic heart disease genesis. Microangiopathy characterized by platelet aggregation and occlusive thrombosis present in small blood vessels of the epicardium and myocardium may cause myonecrosis and focal degeneration with inflammatory infiltrates, and may contribute to the apical aneurysm and cardiomyopathy development, as proposed by Rossi and collaborators. Tanowitz and collaborators showed that microvascular changes are characterized by microthrombi and endothelial cell dysfunction, which are associated with fibrosis and cardiac myocytolysis. Thus, as reviewed by Higuchi and collaborators, the microvascular alterations could be a consequence of the vasodilating substances action as a result of the inflammatory process or the presence of the parasite, leading to ischemic processes that would contribute to the Chagas’ heart disease electrical alterations characteristic.
The hypothesis that the parasite was directly responsible for the lesions occurring during the chagasic infection chronic phase was put aside for years due to the hypothesis that autoimmune mechanisms would be primarily responsible for chronic chagasic myocarditis, as reviewed above. However, in the early 1990s studies, such as those by Jones and collaborators and Higuchi and collaborators, already demonstrated a correlation between the parasite DNA (PCR) or its antigens (immunohistochemistry) persistence and the CD8+ T cells presence in the inflammatory infiltrate in the cardiac tissue of chronic Chagas disease patients, suggesting the parasite direct participation in the development of heart disease myocarditis and severity. Similar results were previously described in experimental models by Younès-Chennoufi and collaborators. These data strongly indicated that chronic Chagas‘ myocarditis would result from immune reactivity to the parasite and its antigens. Tarleton and Zhang, using heart transplantation murine models into the infected animals ear, similar to those described by Ribeiro dos Santos and collaborators, showed that the parasite persistence in muscle tissue is necessary and sufficient for the chronic Chagas’ myocarditis maintenance, this being one of the factors that would determine the Chagas’ disease severity (Figure 2).
A recent study by Fuenmayor and collaborators with endomyocardial fragments obtained from patients with acute chagasic myocarditis showed that 58% of the biopsies had T. cruzi antigens detectable by immunohistochemistry, with the parasite presence not being directly related to inflammatory lesion in the heart tissue the presence, reopening the discussion about the parasite direct participation in inflammatory processes.
In 2003, Higuchi and collaborators suggested that depending on the specific immune response to parasite antigens developed in the Chagas’ infection acute phase, the individual may present the indeterminate chronic form or evolve to severe chronic forms. Thus, an efficient and regulated immune response against the parasite would control parasitemia levels and limit tissue damage; this would be the case in patients presenting the indeterminate form. On the other hand, an inefficient, hyper-responsive, or deregulated response to control the parasite would promote a persistent inflammatory reaction, resulting in a more severe disease, as observed in patients, for example, with chronic cardiomyopathy, associated with myocarditis with fibrosis, extracellular matrix and microcirculation alterations, originating arrhythmias or sudden death, and also leading to ventricular dilatation and heart failure. Thus, the authors propose that the parasite/host interaction during the acute phase would be essential in determining the evolutionary course of the Chagas disease chronic phase.
Recently, it was proposed by Perez-Fuentes and collaborators and Lannes-Vieira that the imbalance between oxidant/antioxidant mechanisms (such as nitric oxide and the enzyme superoxide dismutase) and pro/anti-inflammatory cytokines (such as TNF and IL-10), as well as the persistence of certain CD8+ T cells sub-populations in cardiac tissue, could influence the severity of the lesions developed by the infected individual, which would be the long-lasting and complex relationship consequence between the parasite and the infected individuals poorly regulated immune response who develop the Chagas disease severe forms.
The nature of the cells present in the inflammatory infiltrates found in the Chagas disease patients heart tissue and, consequently, their biological function is still an open question. Histological studies developed in autopsy material performed by Vianna, and later by Köberle, showed the mononuclear and polymorphonuclear cells presence, mainly chronic Chagas disease patients neutrophils and eosinophils in the heart tissue. Regarding eosinophils, Molina and Kierszenbaum observed a direct correlation between the number of these cells and the lesions intensity, suggesting that they might be playing an important role in the cardiac lesions development in patients with Chagas’ infection. However, in recent decades the role and differential contribution of these cells have not been demonstrated.
The resumption of interest in the cells composing Chagas‘ myocarditis occurred in the early 1990s. Reis and collaborators and Higuchi and collaborators showed that in chronic heart disease patients, myocarditis is mainly formed by CD8+ T cells, followed by CD4+, although macrophages, B lymphocytes, NK cells, and polymorphonuclear cells may also be present in smaller numbers. D’Avila Reis and collaborators had shown that CD8+ T cells express CD3, granzyme A, but do not express CD57, suggesting that these are cytolytic T cells and not NK (natural killer) cells. Reis and collaborators and Benvenuti and collaborators also showed the presence of activated macrophage-like cells secreting TNF (tumor necrosis factor) and few B and NK cells associated to the cardiac lesion. There was also an increased expression of MHC (major histocompatibility complex) class I molecules in myocardial cells from Chagas disease patients with chronic myocardiopathy. Tostes Junior and collaborators demonstrated that the contact of CD8+ cells with destroyed myocardial cells in biopsies from chronic Chagas disease patients is evidence that myocardiocyte injury is mediated by cytotoxic T lymphocytes. In this regard, Higuchi and collaborators showed in myocardial lesions from chronic Chagas disease carriers the CD8+ cells presence around areas containing T. cruzi antigens, whereas CD4+ cells were more dispersed throughout the tissue. A recent study by Fuenmayor and collaborators showed no significant differences between CD4+ and CD8+ sub-populations in acute myocarditis in Chagas disease patients.
Studies by Tarleton and collaborators and Rottenberg and collaborators in models of experimental Chagas’ infection have shown the participation not only of CD8+ T cells, but also of CD4+ T cells and B cells, in increasing survival as well as controlling parasitism. CD4+ T cells direct and potentiate effector mechanisms, including the antibody isotypes switching and activation of phagocytes and CD8+ T cells. The latter are able to recognize the infected cells and destroy them, constituting an important effector mechanism in the parasitism control. Cunha-Netto and collaborators studying cells isolated from cardiac biopsies propose that in the Chagas disease chronic phase, CD4+ cells respond in a deleterious way against their own antigens, triggering autoimmune nature inflammatory reactions, which are responsible for the intense chronic myocarditis maintenance.
CD8+ T cells are activated mainly through MHC I molecules and co-receptors expressed by antigen-presenting cells containing T. cruzi remnants. The scarce response of CD4+ T cells in the T. cruzi antigens presence suggests that these antigens presentation through class II MCH molecules is inhibited, as proposed by Tostes and collaborators and Reis and collaborators. However, experimental evidence from Lopes and collaborators suggests that CD4+ T lymphocyte depletion in the Chagas disease chronic phase is correlated with apoptosis-programmed cell death. Host cell integrity may represent a valuable prerequisite for this pathogen survival and development. Pathogen-induced modulation of programmed cell death in the host can result in the elimination of key immune cells or evasion of the parasite from host defenses that could act to limit infection.
Regarding the CD4+ and CD8+ cell sub-populations functional role, studies by Russo and collaborators with depletion of these populations in C3H/HeJ infected with the CL strain showed that CD4+ cells depletion induces increased parasitism in the heart and liver. On the other hand, when treatment with anti-CD8 monoclonal antibodies was performed, no increase in parasitism was observed in the heart tissue, but a marked increase in parasitism in the liver, suggesting that T cell activity against T. cruzi varies according to the target tissue of infection. Tarleton and collaborators showed that mice CD4+ or CD8+ cells depleted show greater parasitism and associated with a slight decrease in inflammation in heart tissue in the acute phase exacerbated in the infection chronic phase. Tarleton and collaborators also showed that animals deficient in b2-microglobulin (b2–/–) when infected with T. cruzi show increased parasitemia and mortality in the infection acute phase with markedly decreased inflammation in skeletal muscle and cardiac tissue, when compared with b2+/+ or b2+/– animals. These experiments suggest that CD4+ and CD8+ T-cell populations, in addition to being necessary for an effective immune response, contribute to the myocarditis establishment.
Cytotoxic CD8 T lymphocytes (CTLs), like NK cells, participate in adaptive and innate immunity, respectively, and use similar mechanisms for the destruction of their “targets”, although there is the distinct receptors involvement and the cytolytic molecule expression regulation, which is constitutive in NK cells but regulated in CTLs. CTLs and NKs can act both by exocytosis release of cytolytic granules constituents, such as perforin and granzymes, and by engagement of cell surface receptors, such as TNF receptor family (TNFR) members, including CD95 (or Fas), as reviewed by Lieberman. Another crucial function of CD8+ T cells is the IFNg (interferon gamma, interferon gamma) production, a cytokine essential for these cells protective activity, as reviewed by Martin and Tarleton. However, recently Leavey and Tarleton showed that CD8+ T cells present in the cardiac tissue of chronically infected animals exhibit an activated/memory phenotype with a CD62L-/lowCD44highprofile, but have attenuated effector function, characterized by low IFN production, when compared to CD8+ cells in the spleen, suggesting the mechanism existence by which T. cruzi persists and generates tissue damage. Thus, the molecular mechanisms that determine the CD8+ T population prevalence in the cardiac tissue of chronic Chagas disease patients remain to be elucidated. Also, it is unclear what the T-cell sub-populations dynamics of activation/migration are, as well as the role they play in the pro- or anti-inflammatory mediators production in the chronic Chagas’ myocarditis formation and perpetuation.
Much remains to be elucidated about the different cell populations roles, such as polymorphonuclear cells, mast cells, dendritic cells, regulatory T cells, and even macrophage and T cell sub-populations, in T. cruzi infection at the systemic level and in the target tissues of infection, especially those that undergo important functional alterations for the chagasic patient, such as the heart tissue.
Data from experimental models have shown that inflammatory cells, such as macrophages and T cells, and the cytokines they produce play important roles in the protective response and immunopathogenesis in parasitic infections. In this context, several studies have shown that cytokines, as well as other inflammation mediators (prostaglandins, thromboxanes, leukotrienes, platelet aggregation factor, etc.), would play an important role in regulating the immune response during infection by T. cruzi, being involved both in resistance to infection and in the mechanisms related to the Chagas disease evolution.
In the T. cruzi acute phase infection, the immunosuppression phenomenon is observed, characterized by the absence of proliferative response to parasite antigens and to other antigens unrelated to T. cruzi. This immunosuppression is attributed to decreased IL-2 production and expression of its receptor (IL-2R), as well as increased suppressor activity of T cells and spleen macrophages, as described by Beltz and collaborators and Tarleton. In this sense, Mosca and collaborators showed that T. cruzi antigens induce proliferative response partial inhibition of peripheral blood mononuclear cells from Chagas disease patients, showing that this process is actively induced by the parasite.
The study in experimental models has greatly contributed to the understanding of the cytokines functional role in T. cruziinfection. Tarleton and collaborators demonstrated that TNF, TGF-b (transforming growth factor), IL (interleukin)-1 and IL-6 are present in the chronically infected animals heart tissue. Powell and collaborators demonstrated that mRNA levels in the myocardium of two mouse strains with resistance and susceptibility profiles do not dichotomize into cytokine types 1 and 2, as initially expected. IFNg, IL-10 and IL-13 have identical levels in the two strains, while the difference arises for IL-4, IL-6 and IL-12, which showed elevated levels in the susceptible mice. Talvani and collaborators and dos Santos and collaborators showed that during C57BL/6 and C3H/He mice acute infection with the T. cruzi Colombian strain there is a predominance of mRNA expression for type 1 cytokines, especially IFNg and IL-12, as well as TNF. On the other hand, IL-4 and IL-10 showed increased expression in cardiac tissue during the infection chronic phase, suggesting regulatory roles for these cytokines in cell-mediated immunity in experimental T. cruzi infection.
Studies in experimental models have also shown an association between IFNg and TNF production and host resistance to T. cruzi infection. It is believed that IFNg associated with TNF activates macrophages, inducing the production of iNOS (inducible nitric oxide synthase), increasing the NO (nitric oxide) production which in turn inhibits the parasite intracellular replication, as described by Gazzinelli and collaborators, Reed and Silva and collaborators. On the other hand, IL-10 and TGF-b inhibit IFNg-induced macrophage activation by inhibiting both NO release and differentiation of IFNg-producing cells, as shown by Silva and collaborators and Abrahamsohn and Coffman. Interestingly, it was shown by Hunter and collaborators that IL-12 administration to T. cruzi infected mice induces increased IFNg and TNF serum levels with reduced parasitemia and increased animals survival rates, but with increased numbers of inflammatory cells associated with parasite nests in cardiac tissue. Still in this sense, Michailowsky and collaborators showed that the administration of IL-12 associated with benzonidazole to animals infected with the T. cruzi Colombian strain, considered drug-resistant, increases the drug inhibitory effect on the parasite, decreasing parasitemia and leading to negative PCR for T. cruzi DNA in blood and heart tissue. However most animals that received IL-12 die with intense myocarditis, suggesting the IL-12-induced cytokines IFNg and TNF play a crucial role in the cardiac inflammation formation.
The TNF participation in the T. cruzi infection pathophysiology is still unresolved. TNF production can be induced directly by the parasite or its antigens, and is dependent on the activation of the transcription factor NF-kB, as shown by Ropert and collaborators. Several T. cruzi-derived molecules, including DNA and the GPI trypomastigotes mucins, stimulate the pro-inflammatory cytokines and chemokines production, involving pathways that depend on the Toll-like receptors (TLR) and the adaptor molecule MyD88, as described by Almeida and Gazzinelli, Shoda and collaborators, Coelho and collaborators, and Campos and collaborators. Aliberti and collaborators showed that TNF, signaling through its receptor p55/TNFR1, plays a critical role in resistance to acute T. cruzi infection by controlling parasite phagocytosis, NO and chemokine production, revealing this cytokine key role in shaping inflammation and controlling parasite growth. On the other hand, Ferreira and collaborators, Pérez-Fuentes and collaborators, and Talvani and collaborators have shown that serum TNF levels correlate with the severity of cardiac dysfunction in chronic Chagas’ disease patients, suggesting that the imbalance in TNF production is directly related to the chronic Chagas’ myocarditis progression.
TGF-b is a cytokine involved in the inflammation control, extracellular matrix components synthesis and deposition, and fibrosis formation. Recent studies by Araújo-Jorge and collaborators have shown that serum levels of TGF-b are increased in Chagas disease patients when compared to non-infected individuals. Higher serum levels have also been observed in heart disease patients, characterized by a lower ventricular ejection fraction, than in individuals with the disease undetermined form. The authors also show that TGF-b levels correlate with fibronectin deposition in the Chagas disease patients heart tissue with cardiac disease. Thus, they suggest that TGF-b plays a role in the Chagas disease pathogenesis.
In T. cruzi experimental models infection, the regulatory cytokines IL-10 and TGF-b are associated with susceptibility to infection by inhibiting IFNg-induced macrophage activation. Endogenous IL-10 neutralization resulted in increased IFNg production and resistance to infection o, as described by Silva and collaborators, Cardillo and collaborators, and Reed and collaborators. In vitro studies with murine and human macrophages showed that TGF-b inhibits IFNg-induced macrophage activation and parasite control. Also, TGF-b favors infection and parasite growth in macrophages. More importantly, treatment of mice with TGF-b in the infection acute phase resulted in increased susceptibility, with increased parasitemia, as described by Silva and collaborators. As described below, TGF-b plays an important role in favoring parasite growth in macrophages that phagocytize apoptotic cells.
IL-4 stimulates B-cell proliferation, regulates allergic reactions, and inhibits macrophage activation. This cytokine is present at high levels in strains of mice susceptible to T. cruzi infection, as shown by Eksi and collaborators and Humphrey and collaborators. However, Wirth and collaborators showed that IL-4 increases T. cruzi phagocytosis and microbicidal activity infected macrophages, by a hitherto unclear mechanism. Michailowsky and collaborators and Soares and collaborators using IL-4 deficient animals infected with the T. cruzi Colombian strain showed that in the infection acute phase IL-4 favors the parasitism establishment, related to mortality. However, in the chronic phase of infection IL-4 is critical for controlling the inflammatory process in cardiac tissue.
The ex vivo peripheral blood mononuclear cells (PBMC) analysis from Chagas disease patients performed by Dutra and collaborators showed that the mRNA expression levels for IL-5, IL-10, IL-13, and IFNg were increased when compared to non-infected individuals. PBMC Stimulation from these patients by parasite antigens (from epimastigotes or trypomastigotes) mRNA revealed increased expression for IFNg and low mRNA expression for IL-10, showing that in Chagas disease carriers pro-inflammatory, anti-inflammatory, and regulatory cytokines co-exist.
Bahia-Oliveira and collaborators reported significantly elevated levels of IFNg in PBMC from trypanocidal drug-treated patients considered cured when compared to treated non-cured patients. Paradoxically, in the untreated group, consisting of patients with the disease chronic form, IFNg was higher in cardiac patients than in asymptomatic patients. Thus, suggesting that this cytokine could be involved in both the chagasic pathogenesis protection and development. Bahia-Oliveira and collaborators suggested the role of IFNg in eliminating the parasite, in conjunction with specific chemotherapy, leading to patients parasitological cure. On the other hand, Gomes and collaborators proposed that in the most Chagas’ heart disease severe forms, IFNg would be involved in the induction of inflammatory response in cardiac tissue. On the other hand, Samudio and collaborators proposed that the disease chronic cardiac form would be associated with increased IL-10 production in response to the parasite, which could even negatively regulate IL-2 production. Studying children infected in the indeterminate form acute phase or early phase, Samudio and collaborators observed a predominant Th1 (IFNg) pattern in the acute phase group, while individuals in the early indeterminate phase had a Th0 pattern (IFNg and IL-4). According to the authors, this cells selective induction with a Th0 pattern would be important for the development of the cellular and humoral immune response that would control the parasite load, thus contributing to less morbidity in the late chronic phase.
The cardiac biopsies studies from chronic Chagas’ disease patients by D’Avila Reis and collaborators showed the TNF presence in cells morphologically characterized as macrophages in the myocardial lesions. Reis and collaborators studying myocardium from 25 patients with chronic heart disease observed the T. cruzi antigen spresence in 68% of the fragments studied. CD8+ cells were the main cells found in the tissues, while CD4+ cells were present in smaller numbers. Cells expressing IL-2 or its receptor were scarce, suggesting that patients with chronic Chagas’ disease may have an immune imbalance with suppression of IL-2 production. The expression of IFNg was intense, with a positive correlation between the cells secreting this cytokine and CD8+ cells number, suggesting that these cells are the main source of this cytokine in the tissues of chagasic heart disease patients. A moderate number of cells secreting IL-4, IL-6 and TNF were also observed, suggesting that these cytokines, and especially IL-4 produced by CD4+ T (Th2) cells, could be related to parasite dissemination.
Cunha-Neto and collaborators and Abel and collaborators when studying endocardial biopsies of chagasic heart disease patients to be submitted to transplantation, showed a predominance of cells producing IFNg and TNF in lesion areas, with results in agreement with the immunohistochemical findings of Reis and collaborators.
More recent studies by Gomes and collaborators show that IFNg production by peripheral CD3+CD4+ cells is directly associated with the cardiomyopathy severity. On the other hand, Laucella and collaborators showed that the frequency of circulating IFNg-producing CD3+CD8+ cells is inversely correlated to the chronic illness and disease severity in patients exposed to reinfection. These seemingly contradictory results should be considered in light of the roles described for this cytokine in experimental models, either controlling parasite growth or favoring inflammation. Gomes and collaborators also showed that IL-10 production by macrophages/monocytes is associated with the development of the Chagas‘ infection indeterminate form.
Finally, the pro-inflammatory cytokines IFNg and TNF could also modulate the expression of other molecules groups, such as cell adhesion molecules and chemokines, involved in the control of T. cruzi growth in macrophages, as well as in the control of cell recruitment and migration, contributing to the inflammation chronification, as recently shown by Aliberti and collaborators, Michailowsky and collaborators and Gomes and collaborators. On the other hand, cytokines with a regulatory profile, such as TGF-b, could contribute to the control of inflammation, but also to the formation of scarring and fibrosis by deposition of extracellular matrix components. Thus, the participation of cytokines in the pathogenesis of chagasic heart disease still deserves many new chapters.
Viana and Chagas demonstrated that there is no correlation between parasitism and inflammatory reaction in chronic chagasic myocarditis. However, Vianna and Milei et al. showed that, in the cardiac tissue of patients with chronic Chagas’ disease, mononuclear infiltrates and extensive areas of fibrosis are common findings. These findings were recently reproduced by Carvalho et al. in the experimental model of chronic infection (over 20 years) of Rhesus monkeys by T. cruzi (Figure 3).
In fact, a correlation between the intensity of the inflammatory reaction and fibrogenesis was described by Andrade et al. in an experimental murine model. In the sub-acute stage of experimental infection of BALB/c mice with the 12SF strain of T. cruzi, Andrade showed deposits of fibronectin, laminin and collagen in the myocardium and perivascular spaces, with fibronectin deposition directly correlated with the presence of inflammatory infiltrates. In the chronic phase, the mice presented deposits of fibronectin, laminin, and type III, pro-III and IV collagens. Also, these authors investigated the effect of chemotherapy on fibrotic and inflammatory changes in mice chronically infected with 21SF and Colombian strains. They demonstrated that treatment in the chronic phase of the infection with the drugs benznidazole and MK-436 (nitroimidazole) results in the regression of inflammation and fibrosis, concluding that not only inflammation, but also fibrotic changes in the extracellular matrix are reversible in dependence. of parasite control. Some recent studies have addressed the nature of fibrogenic factors in T. cruzi infection. Pinho et al. showed that antigens released by T. cruzi bind to fibroblasts and muscle cells and induce increased expression of extracellular matrix components, such as fibronectin, laminin, and type I collagen, which may contribute to the formation of cardiac fibrosis. Marino et al. showed that inflammatory cells, predominantly CD8+, found in the heart tissue of animals infected with T. cruzi VLA4 expression (very late antigen 4, CD49d, α4b1) https://en.wikipedia.org/wiki/CD49d and are surrounded by a fine network of fibronectin (Figure 4).
The authors proposed that cytokines produced by these cells could contribute to the production of extracellular matrix components and these could contribute to the anchoring of cells and cytokines, migration and activation of inflammatory cells, perpetuating the process that would result in chronic heart disease. More recently, Araújo-Jorge et al. showed a correlation between serum levels of TGF-b and cardiac fibrosis in patients with heart disease with reduced ventricular ejection fraction, reinforcing the role of fibrosis in the pathogenesis of Chagas’ heart disease.
Apoptosis-programmed cell death of immune cells, including T and B cells, occurs during the course of T. cruzi infection. In infected experimental models, there is a significant loss of CD4+ T-cells by increased expression of Fas (CD95) and Fas-ligand (CD95L), with subsequent induction of apoptosis by activation-induced cell death, as shown by Lopes et al. T. cruzi-infected mouse fibroblasts are not induced to apoptosis, as described by Clark and Kuhn. On the other hand, cardiomyocytes undergo apoptosis during T. cruzi invasion both in vitro and in vivo, with the rate and speed of death being related to the parasite strain, occurring earlier and at higher levels during interaction with parasites of the type T. cruzi I, as described by de Souza et al. However, many molecular aspects involved in the induction of apoptosis as a result of the cardiomyocyte/T. cruzineed to be addressed in order to understand the contribution of this process to the pathogenesis of chagasic heart disease.
A study by Freire de Lima et al. showed that phagocytosis of apoptotic cells by infected macrophages results in the production of prostaglandin E and TGF-b, with an increase in the number of parasites. Also, the injection of apoptotic cells in infected animals results in an increase in parasitemia, showing that the induction of apoptosis and the phagocytosis of apoptotic cells can be an important immunoregulatory mechanism, as well as the escape and perpetuation of T. cruzi in the host.
The predominance of CD8+ T-cells in the myocardium of patients in the chronic phase of Chagas’ disease has been described by several authors, including D’Avila Reis et al, Higuchi et al, and Tostes et al. However, the molecular mechanisms that determine the prevalence of CD8+ cells in this cardiac tissue remain unclear. Most inflammatory cells in the myocardium of patients with chronic Chagas’ disease express cell adhesion molecules such as LFA-1 (leukocyte function-associated antigen-1; CD11a/CD18) ligand of ICAM-1 (intercellular adhesion molecule-1; CD54), CD44 fibronectin and hyaluronic acid ligand and VLA-4 (very late antigen-4; CD49d/CD29, α4β1) ligand of VCAM-1 (vascular cell adhesion molecule-1; CD106), and fibronectin. This result led D’Avila Reis et al. to suggest that these molecules would contribute to the progression of the inflammatory reaction by mediating the adhesion of lymphocytes to the endothelium of cardiac tissue vessels activated by cytokines and by being important in the infiltration and localization of cells in inflammatory sites. Laucella et al. detected a greater number of VLA-4+ cells among PBMC of patients with Chagas’ disease with severe heart disease compared with those with less severe heart disease.
Using C3H/HeJ mouse models infected with the Colombian strain of T. cruzi, dos Santos et al. and Marino et al. showed the prevalence of CD8+ T lymphocytes in the cardiac tissue of chronically infected animals. In this model, both CD4+ and CD8+ T-cells in cardiac tissue express an activation phenotype, which translates into low expression of L-selectin (CD62L) and high expression of LFA-1 and VLA-4 (CD62Llow, LFA-1high, VLA-4high), which would potentially allow the interaction of these inflammatory cells with activated endothelium expressing VCAM-1 and ICAM-1.
Laucella et al. showed the presence of high levels of VCAM-1 in the serum of patients with acute and chronic Chagas’ disease, while ICAM-1 and CD44 have high levels only in the acute phase, showing a decrease in the chronic phase of the infection. Only serum levels of P-selectin (CD62P) were associated with disease severity. The release of the molecules is affected by the same stimuli that increase their expression on the cell surface, such as IFNg and TNF. In this context, Benvenuti et al. showed that severe chronic chagasic heart disease is characterized by increased expression of both ICAM-1 and VCAM-1 in cardiac vascular endothelium, induction of class I MHC molecules in cardiomyocytes, and by a predominance of CD8+ cells in inflammatory infiltrates. Interestingly, the study by Marino et al in infected mice showed that the kinetics of ICAM-1 and VCAM-1 appearance on the endothelium of blood vessels of cardiac tissue coincides with the kinetics of entry of inflammatory cells into this tissue. In addition, dos Santos et al. revealed that a high proportion of circulating CD8+ T-cells, compared to CD4+, express LFA-1 and VLA-4, which could facilitate the preferential migration and predominance of these cells in the myocardium of individuals chronically infected with T. cruzi. The participation of IFNg in the modulation of ICAM-1 expression and the participation of this molecule in cell migration in T. cruzi infection and formation of cardiac inflammatory infiltrate was confirmed by Michailowsky et al. The authors also showed that the high susceptibility of animals genetically deficient in ICAM-1 to T. cruzi infection is associated with a decrease in the number of CD4+ and CD8+ T-cells in cardiac tissue.
These data show that cell adhesion molecules participate in the process of migration and formation of the cellular infiltrate involved in the immune response that controls the parasite, also suggesting that these molecules participate in the formation of chronic inflammation in cardiac tissue during T. cruzi. It remains to be clarified whether there is a differential role of these molecules in protective immunity and in the formation of chronic inflammation related to the severity of chagasic heart disease.
Chemokines form a superfamily of small proteins (8-10 kD) that play an important role in immune and inflammatory reactions. As reviewed by Luster, Proudfoot and Mantovani, based on the presence of cysteine residues, which may or may not be intercalated by non-conserved amino acid(s), represented by X, CXC(a), CC(b), C(g) and CXXXC(d) families of chemokines were identified. These molecules interact with a family of receptors with 7 trans-membrane domains in serpentine conformation linked to the G protein. In general, CXC chemokines act on neutrophils and T- and B-cells, while CC chemokines act on a broader spectrum. of cell populations, such as monocytes, basophils, eosinophils, T-cells, dendritic cells and natural killer cells, not acting on neutrophils. Probably all cell types, such as endothelial cells, cardiomyocytes, fibroblasts, megakaryocytes, T-cells, macrophages, etc., when stimulated appropriately and depending on the nature of the stimulus, produce several types of chemokines simultaneously.
Chemokines can bind to various molecules found in the extracellular matrix with affinities ranging from nanomolar to millimolar, as described by Fadden and Kelvin. They are basic proteins and bind avidly to heparin and heparan sulfate (negatively charged). Heparan sulfate and proteoglycans retain chemokines in the extracellular matrix and on the surface of endothelial cells, a process that can serve to establish a local concentration gradient from the cellular source of chemokine secretion, as well as stabilize these molecules by increasing their biological action time, as reviewed by Luster.
There are several biological functions of chemokines, including (i) induction of adhesion to the endothelium and to extracellular matrix components, (ii) chemotactic migration of leukocytes, (iii) control of angiogenesis, (iv) control of the production of extracellular matrix components, (v) regulation of growth, activation and differentiation (including Th1/Th2 impairment) of lymphocytes, (vi) release of enzymes from intracellular reserves, (viii) formation of oxygen radicals, (ix) alteration of the cytoskeleton, (x) generation of lipid mediators of intracellular activation signals, (xi) proliferation of hematopoietic precursors, and (xii) control of invasion and multiplication of pathogens. These effects may be important in mediating host resistance to pathogens and in the immunopathogenesis of various diseases, including, for example, infectious and autoimmune diseases.
The inflammatory reactions observed both during the acute and chronic phases of T. cruzi infection seem to play an important role in the pathogenesis of Chagas’ disease. In the affected tissues, local production of several immunological mediators occurs, causing an intense migration of leukocytes during the interaction between the parasite and the host cells. The quest to understand these processes led to the study of the expression and function of chemokines and their receptors in T. cruziinfection in patients with Chagas’ disease and in experimental models.
The action of CC or b-chemokines seems to be particularly beneficial to the host during T. cruzi infection. Lima et al. and Villalta et al. showed that human macrophages infected in vitro by T. cruzi produce CC-chemokines. They also showed that the addition of CCL3/MIP-1α, CCL4/MIP-1b or CCL5/RANTES to infected macrophages results in intracellular destruction of trypomastigotes by a NO-dependent mechanism. These findings were confirmed in mouse inflammatory macrophages by Aliberti et al. Machado et al. demonstrated that cardiomyocytes from in vitro cultured mice infected with T. cruzi produce chemokines, iNOS, and NO, which could contribute both to the control of the parasite, but also to the pathogenesis of chagasic heart disease.
Interestingly, Machado et al. showed that the treatment of cardiomyocytes infected with T. cruzi with TNF results in induction or increased expression of CC-chemokines. Also, Aliberti et al., macrophages infected and treated with TNF or IFNγ express more CC-chemokines, while treatment with IL-10 or TGF-β leads to reduced production of these chemokines, suggesting that pro-inflammatory and regulatory cytokines can control the parasitism and the formation of inflammation by chemokine-dependent mechanisms.
In different murine models of acute and chronic T. cruzi infection, increased mRNA levels of several chemokines (CCL2/MCP-1, CCL3/MIP1α, CCL4/MIP-1β, CCL5/RANTES, CXCL9/Mig and CXCL10/IP -10) was observed in cardiac tissue associated with the formation of the inflammatory infiltrate, as shown by Talvani et al., Aliberti et al., dos Santos et al., and Marino et al. Petray and colleagues using neutralizing antibodies showed that the CCL3/MIP-1α chemokine is involved in macrophage recruitment during acute infection. However, the role of chemokines in the differential migration of cell populations in T. cruziinfection needs to be further explored. In a recent study, Roffê et al. showed that immunization of animals with the DNA vaccine expressing CCL4/MIP-1b led to the production of antibodies that were related to the exacerbation of inflammation and cardiac fibrosis, without altering the parasitism, revealing that this chemokine is related to the control of excessive inflammation and pathogenesis in T. cruzi infection.
Plasma concentrations of chemokines have been correlated with worsening of the disease in patients with heart failure. In this sense, Talvani et al. found that high plasma levels of the chemokine CCL2/MCP-1, but not of CCL3/MIP1α, are directly correlated with cardiac damage in patients with Chagas’ disease and heart disease.
The evaluation of the expression of chemokine receptors on the surface of mononuclear cells from peripheral blood of patients with Chagas’ disease performed by Talvani et al. showed a higher frequency of cells expressing CCR5 and CXCR4 in patients with Chagas’ disease compared to non-infected individuals. In addition, cells isolated from patients with severe heart disease had a lower frequency of cells expressing CXCR4. Gene polymorphism studies of CCR5 59029A/G performed by Calzada et al in Peruvian patients and Fernandez-Mestre et al in Venezuelan patients revealed that the G allele, which results in lower expression of CCR5, is found more frequently in asymptomatic patients than in cardiac patients. Recently, Gomes et al. observed that peripheral blood mononuclear cells from cardiac patients stimulated in vitro with T. cruzi antigens showed an increase in the percentage of CD4+ and CD8+ T lymphocytes co-expressing CCR5/IFNg, CXCR3/IFNg and CXCR3/TNF. Interestingly, asymptomatic patients had an increased percentage of CD4+ and CD8+ T-lymphocytes co-expressing CCR3/IL-10 and CCR3/IL-4. Therefore, it is possible that the genesis of chronic chagasic myocarditis is related to the migration of cell populations with different patterns regarding the expression of chemokine receptors and effector capacity of peripheral blood cells to the heart, as proposed by Lannes-Vieira and Marino et al.
Marino et al. observed a predominance of CD8+ T-lymphocytes in the cardiac tissue of C3H/He mice infected with the Colombian strain, in relation to CD4 T-cells, expressing CCR5. Aiming to modulate cardiac inflammation, mice were treated with Met-RANTES (N-terminal-methionylated RANTES), a selective antagonist of CCR1 and CCR5 receptors. Interestingly, the authors observed that the drug did not interfere with parasitism, but significantly reduced the number of CD4 and CD8 CCR5+ T-lymphocytes in cardiac tissue and the deposition of fibronectin, resulting in increased survival of treated animals. Thus, these data strongly suggest that CCR5+ cells are not crucial for parasite control, but play an important role in the pathogenesis of chagasic heart disease. More recently, Machado and collaborators confirmed the predominance of CD8+CCR5+ cells in the cardiac tissue of animals infected with T. cruzi using the C57BL/6 model infected with the Y strain, showing that the process results from T. cruzi infection, and is not a finding of a particular model. Furthermore, the authors showed that CCR5 expression was increased in CD8+ T-cells by in vitro stimulation with T. cruzi antigens, showing the central role of parasite persistence. In agreement with the data of Marino et al., Hardison et al., using mice genetically deficient in CCR5, observed, particularly in the initial phase of infection, a marked decrease in the migration of macrophages and T-cells to the heart. However, the authors showed that the total absence of CCR5 resulted in high parasitemia and cardiac parasitism. Taken together, these data suggest that CCR5 plays an important role in the influx of inflammatory cells into cardiac tissue, which may be influenced by host genetic factors, and that different subpopulations of CCR5+ cells may play different roles in parasitism control and in the immunoregulation of chagasic myocarditis, which needs to be further explored.
It is estimated that susceptibility to infectious diseases occurs in a small percentage, ranging from 0.1% to 10%, of the population exposed to infectious agents. The progression of an infection, as well as the development of different clinical forms and different degrees of severity, is related to the complex parasite/host relationship, which also involves environmental factors (nutritional status, previous immune status, such as exposure to other pathogens), and the genetic characteristics of the pathogen and the host. In the case of T. cruzi infection, the spectrum of expression of Chagas’ disease varies from asymptomatic patients to patients with heart disease with severe heart failure or the digestive form with formation of megas. These would be strong evidences of the influence of genetic factors on susceptibility to infection by T. cruzi.
Considering the fundamental role of MHC class II molecules in the process of antigenic presentation and control of intracellular microorganisms, Fernandez-Mestre and collaborators carried out a study of gene polymorphism of DRB1 and DQB1 molecules in Venezuelan Chagas’ disease carriers. These have a decreased frequency of the DRB1*14 and DQB1*0303 alleles in relation to uninfected individuals, suggesting the independent protective role of these molecules against chronic infection. The study of patients with or without heart disease revealed a higher frequency of the DRB1*01, DRB1*08 and DQB1*0501 alleles and a lower frequency of DRB1*1501 in patients with arrhythmia and congestive heart failure. These data suggest that HLA class II genes may be associated with the development of chronic infection and chronic damage to heart tissue.
The diverse findings regarding the differential expression of cytokines, chemokines, chemokine receptors and effector factors in the acute and chronic phases of Chagas’ disease, in patients with different clinical forms and in experimental models made these molecules targets of initial studies of gene polymorphism in Chagas’ disease.
Serum levels of TNF were related to the evolution of the disease and the severity of chagasic heart disease by Ferreira et al., Peréz-Fuentes et al., and Talvani et al., suggesting the participation of TNF in the formation of chronic inflammation and cardiac injury. The most frequently performed gene polymorphism studies involve single-base nucleotide polymorphism (SNP) frequency analysis. The study by Beraun et al. on the frequency of TNF promoter polymorphisms (-308, -244, and -238) in a small number of Peruvian patients showed no association when comparing asymptomatic and cardiac patients. The study in Mexican patients carried out by Rodríguez-Pérez et al. showed a higher frequency of the TNF-308A polymorphism, which results in a transcription 2 times greater than the TNF-308G allele, in patients with Chagas’ disease when comparing them with individuals not infected by T. cruzi. This study also revealed a higher frequency of this polymorphism in patients with heart disease when compared to asymptomatic patients. Drigo et al., studying the frequency of the TNF-308A allele and the microsatellite TNFa2 allele, which determine high TNF production, in 42 Brazilian Chagas’ disease patients with ventricular ejection fraction ≤ 40% showed that patients expressing these alleles have lower survival time compared to patients carrying other alleles. However, recently Drigo et al. analyzed these same alleles in relation to the progression of chagasic heart disease in 160 patients with heart disease and 80 asymptomatic patients matched for age and geographic area. Patients with heart disease were grouped according to left ventricular dysfunction in severe, moderate and without cardiac dysfunction. The results indicate that TNF-308A and TNFa2 polymorphisms are not associated with the development of heart disease, nor with progression to severe forms in these patients. In the same line of research, Ramaswamy et al. showed that in relation to the BAT-1 gene, the nt -22 and nt -48 variants, which are really effective in modulating the expression of TNF and IL-6, are associated with susceptibility to chagasic heart disease. These data suggest that these variants, proposed to result in less efficient control of the production of pro-inflammatory cytokines, may contribute to the high production of these cytokines in patients with Chagas’ disease with chronic heart disease.
Conceptually, the differential expression of molecules that participate in macrophage effector mechanisms can significantly contribute to the outcome of parasite control and progression of Chagas’ disease. Polymorphism studies of the NRAMP1 gene (natural resistance-associated macrophage protein 1) and the iNOS/NOS2 promoter carried out in Peruvian patients by Calzada et al. non-infected controls and carriers of Chagas’ disease, as well as indicating that the polymorphisms of these molecules are not related to the pathogenesis of chagasic heart disease. However, the possibility of participation of these molecules directly or indirectly in cardiac injuries should be considered, for example, in the case of deregulation of iNOS production and long-term production of high amounts of NO in cardiac tissue.
Several studies have shown that the infection of macrophages and cardiomyocytes results in the production of the CC-chemokines CCL2/MCP-1, CCL3/MIP-1a, CCL4/MIP-1b and CCL5/RANTES. Studies of CCR5 polymorphism (CCL3, CCL4 and CCL5 receptor) in patients with Chagas’ disease carried out by Calzada et al and by Fernandez-Mestre et al revealed that the CCR5-59029G allele, associated with low CCR5 expression, is more frequent in asymptomatic patients than in patients with heart disease. Recent studies by Marino et al. showed that CCR5 expression is increased in experimental T. cruziinfection in mice, and this molecule is involved in the influx of inflammatory cells into cardiac tissue. Interestingly, Gomes et al. showed a higher frequency of CCR5+ cells expressing IFN in cardiac patients than in asymptomatic patients. Taken together, these data suggest that the CCR5 polymorphism that results in higher expression of CCR5 may be associated with the development of the severe form of chagasic heart disease; however, studies in Brazilian patients and with appropriate samples (number and matched by age and geographic area) should be performed.
Together, the data presented above suggest the participation of molecules involved in antigen presentation and parasite control, in the cell migration process and in the regulation of the immune response in the pathogenesis of chagasic heart disease. These findings also point to the fact that an immune response with the appropriate focus and intensity can control the spread of T. cruzi in the absence of disease, as in most patients. Thus, the understanding of the molecular events involved in the formation of inflammation can contribute to the development of new therapeutic strategies that result in the control of the parasite and the inflammation that leads to chronic cardiac dysfunction, with the establishment of the homeostatic balance in the T. cruzi/host and better prognosis for patients with chronic Chagas’ disease.
Mariana Caldas Waghabi
Laboratory of Functional Genomics and Bioinformatics, Instituto Oswaldo Cruz/Fiocruz
The evolution of the clinical cardiac forms of Chagas’ disease is well characterized through clinical and experimental studies, being classified into acute form, indeterminate form and chronic chagasic heart disease, which includes chagasic cardiomyopathy and the arrhythmogenic form of the disease, conduction disorders ventricular and/or intraventricular atrium with preserved or slightly altered ventricular function.
In the acute phase of Chagas’ disease, in most symptomatic cases, it is observed fever, muscle pain, irritability, conjunctivitis, anorexia, vomiting, diarrhea, lymphadenopathy, hepatosplenomegaly, myocarditis (Figure 1), anemia, thrombocytopenia, leukocytosis with lymphocyte predominance, abnormal liver function, and elevated levels of cardiac enzymes. As a result of this infection, the host develops a specific immune response after a few weeks, without, however, the parasites being completely eradicated. In any case, this strong and specific immune response associated with the innate immune response controls the acute infection progressing to the chronic phase. Chagasic cardiomyopathy is still an incurable disease and, according to PAHO data, it represents one of the main causes of myocardial diseases in Latin America. Cardiac involvement occurs in approximately 80 to 90% of acute cases, and is clinically characterized by acute myocarditis with sinus tachycardia, changes in repolarization and low voltage on the electrocardiogram, dilation of ventricular cavities, systolic dysfunction, pericardial effusion, and signs of heart failure. Histopathological analysis reveals focal myocytolysis, necrosis, and large areas of inflammation. In the presence of acute myocarditis, endomyocardial biopsy may allow visualization of the amastigote forms of the parasite, through Giemsa staining, or through specific immunohistochemistry for some parasite antigens. The inflammatory infiltrate initially composed of polymorphonuclear cells is gradually and predominantly replaced by mononuclear cells, being frequently associated with nests of parasites and/or parasitic antigens. Despite the good evolution of most patients in the acute phase of the disease and subpatent parasitemia, there is no proof of cure of the disease with eradication of the parasite.
Approximately 70% of infected individuals will not develop clinical disease and will remain in the so-called indeterminate form of the disease and have a good long-term prognosis. About 30% of infected individuals develop the clinical symptoms of the chronic phase of Chagas’ disease, which include digestive, cardio-digestive, neurological, and cardiac manifestations, the latter being the most significant form due to the observed frequency and severity. Chronic cardiomyopathy (Figure 2) is one of the main manifestations associated with morbidity in Chagas’ disease, possibly triggered by the parasite-host interaction that occurs during the acute phase. It is clinically evident and can include apical aneurysms, severe biventricular dysfunction, progressive heart failure, severe atrioventricular and intraventricular conduction disorders, complex ventricular arrhythmias, thromboembolic phenomena, and cardiomegaly with high rates of morbidity and mortality, either from myocardial failure or sudden death. Sudden death, which can occur even in asymptomatic patients, represents the main cause of death in patients with chagasic cardiomyopathy, and chagasic heart failure etiology is associated with higher mortality than heart failure from other causes. On the electrocardiogram, the most common anomalies are intraventricular block of the right bundle branch, left anterior fascicular block, extrasystoles, changes in Q-waves and increased QRS and QTc intervals. Histologically, there is diffuse lymphocytic myocarditis, few parasite nests and diffuse interstitial fibrosis. Often the first manifestation of chagasic cardiomyopathy can be sudden death or pulmonary or systemic thromboembolic phenomena. Fibrosis (Figure 3) is one of the most significant manifestations of chronic chagasic heart disease and is associated with inflammatory infiltrates and degenerating cardiomyocytes.
Fibrogenesis can be defined as the production of extracellular matrix components mainly by fibroblasts. It occurs during the physiological process of tissue remodeling or can be induced after tissue damage, caused, for example, by pathological agents. The tissue repair process is among the natural mechanisms of protection of organisms, however, when the injurious stimulus is maintained for prolonged periods, there is a loss of balance between the production and degradation of the components of the extracellular matrix, leading to the gradual replacement of the tissue. functional by a connective tissue. Excess production and deposition of extracellular matrix components gives rise to the process called fibrosis, which is triggered in numerous pathologies, including Chagas’ disease. The mechanisms that regulate progressive fibrosis in different organs and tissues are similar and have common characteristics, such as the progressive accumulation of connective tissue and excessive collagen deposition, resulting in the replacement of normal tissue architecture and impairment of its functional activity. This process is mediated by cytokines and soluble growth factors that regulate cell migration, proliferation and differentiation, as well as the synthesis and degradation of extracellular matrix components.
The development of fibrous tissue can occur in the presence or absence of parenchymal cells. Reparative or scarring fibrosis is an adaptation to parenchymal loss and is crucial for preventing structural tissue destruction. The extent of fibrous tissue is inversely proportional to the regenerative capacity of parenchymal cells. After necrosis damage, as occurs in infarction, myocardial tissue responds with inflammation and repair. During this process, the necrotic tissue is replaced by granulation tissue that becomes macroscopically visible. Cardiomyocytes are strictly differentiated with extremely reduced proliferation capacity. This characteristic leads to restrictions on regenerative growth in situations of increased demand and/or cell loss, being partially offset by the global increase in its cell size and structure, which can lead to progressive dysfunction and heart failure. In cardiac fibrosis, fibrillar collagen occupies the space of lost cells, serving as replacement tissue. In addition to this collagen deposition at the site of damaged tissue, as a healing response (reparative fibrosis), there is also an increase in collagen deposition distal to the area of infarction (reactive fibrosis).
TGF-β (transforming growth factor-β) is one of the main cytokines involved in the regulation of extracellular matrix formation and degradation. TGF-β participates in the regulation of fibrosis by stimulating (1) fibroblast chemotaxis, (2) the transformation of fibroblasts into myofibroblasts, (3) the epithelial-mesenchymal transition, (4) the production of extracellular matrix components; such as fibronectin, laminin, collagen, vitronectin and thrombospondin, (5) inhibition of metalloprotease synthesis and (6) production of tissue inhibitors of metalloproteases (TIMPs). The production of TGF-β in areas close to tissue damage may contribute to myocyte hypertrophy and the process of deposition of extracellular matrix proteins.
Inhibition of matrix degradation is due to decreased pericellular proteolytic activity with increased expression of TIMPs. When administered in vivo, TGF-β can induce fibrosis at the site of administration. In the liver, it plays an important role in the pathophysiology of hepatic fibrogenesis. In transgenic mice with hepatic overexpression of active TGF-β, multiple tissue damage is observed, including hepatic fibrosis and extensive glomerulonephritis. Other important cellular functions including differentiation, adhesion, cell migration and immune response are also controlled by this mediator.
In addition to this cytokine, others also participate in this concert of biological activities involved in the genesis and regulation of fibrosis, including (i) pro-fibrogenic cytokines, such as tumor necrosis factor (TNF), endothelin, and platelet-derived growth factor ( PDFG), basic fibroblast growth factor (bFGF), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1-alpha (MIP-1a), interleukin 1 (IL-1), interleukin 5 (IL-5), interleukin 8 (IL-8) and interleukin 13 (IL-13) and (ii) anti-fibrogens such as interferon gamma (IFNg).
Among the pro-fibrogenic molecules, we highlight endothelin, which is a vasoconstrictor peptide of 21 amino acids associated with fibrosis in different tissues, including the heart, where it induces an increase in collagen synthesis by fibroblasts. Treatment with cellular endothelin receptor antagonists reduces myocardial fibrosis in mice with hypertension. There are reports of the interaction between several signaling systems present in cardiac tissue including endothelin, TGF-β and adenosine triphosphate (ATP) via purinergic receptors. TNF overexpression in transgenic mice induces ventricular hypertrophy by increasing collagen deposition in the myocardium, this effect being attenuated by treatment with anti-TNF antibody, suggesting that this pro-inflammatory cytokine has a critical role in extracellular matrix remodeling, and may be a potential target for immunotherapy. The anti-fibrogenic action of IFNg is of particular interest because of its dual role in the fibrosis process, which can stimulate macrophages to produce fibrogenic cytokines and at the same time inhibit collagen gene expression and fibroblast proliferation.
Evidences show that Trypanosoma cruzi interacts with components of the host’s extracellular matrix, producing the lysis of products that play an important role in the mobilization of the parasite and its infectivity. The increased expression of extracellular matrix components in cardiac tissues of patients with chronic Chagas’ disease has already been described (Figure 4). Matrix components can adsorb parasite antigens and cytokines that can contribute to the establishment and perpetuation of inflammation. In 2002, Pinho et al. demonstrated that the adsorption of parasite antigens by sensitized cells led to an increase in the expression of matrix components such as fibronectin, laminin and type-I collagen. This data reinforces the idea that antigens released by T. cruzi may be involved in the establishment of inflammation, sensitizing uninfected host cells, leading to an immune response against parasite antigens. Some chemokines have already been characterized as involved in the pathogenic development of chagasic myocarditis. Among these, we highlight the increase in the expression of CCL5/RANTES and CCL3/MIP-1alpha, and its receptor CCR5, in the heart of mice infected by T. cruzi, suggesting a role of CC-chemokines in myocarditis triggered by T. cruzi.
In the heart, in addition to fibroblasts, cardiomyocytes also secrete fibrogenic cytokines such as TGF-β and FGF. These cytokines associated with those produced by inflammatory cells recruited to the inflamed tissue may contribute to triggering and maintaining the fibrotic process. In addition to the high expression of collagen during the chronic phase of Chagas’ disease, other components of the extracellular matrix also have their expression increased during the evolution of the fibrosis process, such as laminin and fibronectin. Furthermore, in response to injury, TGF-β promotes the migration and proliferation of fibroblasts required for tissue regeneration.
The relationship between high levels of TGF-β and cardiac fibrosis in patients with Chagas’ disease and in experimental models may be related to studies in which an increase in protein and mRNA expression for TGF-β and its cellular receptors is also observed in myocardial infarcted regions in response to healing. This increase in mRNA expression for TGF-β 1 in mice induces an elevated expression of CTGF (connective tissue growth factor) in the infarcted tissue. The cells likely responsible for the production of CTGF are myofibroblasts and fibroblasts. Increased CTGF expression is associated with mRNA expression for collagen, indicating that CTGF plays a significant role in the pathological development of infarction.Plasma levels of TGF-β in patients with chronic heart disease vary significantly, rising from 0.44 ng/mL in uninfected individuals to 21.4 ng/mL in chronic patients. Although all patients with Chagas’ disease show positive variations in TGF-β levels, a trend towards higher levels is observed in patients with more severe manifestations of cardiac dysfunction, as evidenced by different clinical parameters, such as electro and/or echocardiogram. More recent studies have not confirmed this correlation, possibly due to the drugs being used by patients. Concomitantly with the high levels of TGF-β in patients with heart disease, immunohistochemical assays show intense cardiac fibrosis, which can be visualized by the increase in the expression of extracellular matrix elements, such as fibronectin, in addition to the strong expression (in the nucleus) of the phosphorylated Smad 2 protein (PS-2) involved in the TGF-β signaling pathway (Figure 5).
Some authors describe that the progressive accumulation of interstitial collagen interferes with the normal conformation of the myocardium, leading to a loss in the synchrony of ventricular contractions during systole, and contributing to a spectrum of ventricular dysfunctions that involve both the systolic and diastolic phases of the cardiac cycle. Myocardial fibrosis is probably involved in the formation of arrhythmias (ventricular tachycardia or fibrillation), major causes of sudden death in patients with chronic Chagas’ disease. The increase in collagen fibers also interferes with the electrical properties of the myocardium. Fibrosis blocks the cardiac impulse, which cycles through an alternative pathway, slowing conduction. Furthermore, the thick collagen septa between the muscle bundles could interfere with the lateral conduction of the electrical impulse, as demonstrated in senescent hearts compared to young subjects.
In addition to fibrosis, changes in the electrical impulse conduction system represent another important manifestation of chagasic cardiomyopathy. Additionally, another possible role of TGF-β could be related to the change in cellular plasticity by the loss of cellular contacts mediated by alteration of junctional proteins, such as connexin. This hypothesis is validated by the fact that in several systems that have already been studied, TGF-β is able to affect intercellular communication mediated by “gap” junctions, which are structural bases of electrical impulse transmission in the heart.
In cardiomyocytes infected by T. cruzi in vitro and in vivo, a strong reduction in connexin-43 (Cx43) labeling can be observed, and the consequent decrease in electrical conduction in these cells; in addition, the direct participation of gap junctions in the alteration of electrical conduction and arrhythmogenesis in the myocardium was also demonstrated; and that TGF-β participates in the disorganization of Cx43 plaques in mouse and rat hearts. Signaling disorders at gap junctions can lead to changes in impulse propagation between cardiomyocytes and generate ventricular arrhythmias in the myopathic heart.
Andrade et al. were the first to describe an experimental murine model for pathogenesis studies, in which they demonstrated, in the acute phase of experimental infection in BALB/c mice with the 12SF strain of T. cruzi, deposition of fibronectin, laminin and collagen in the myocardium and in perivascular spaces, bordering areas of inflammatory infiltrates.
The pathogenic mechanisms involved in chagasic heart disease are complex and have not yet been fully elucidated. It is believed that the parasite plays a key role during the acute and chronic phases of the disease, exerting an immunosuppressive effect, causing tissue damage, acting directly on infected cells and indirectly by inducing the development of an autoimmune phenomenon and hypersensitivity. In this way, this process favors the persistence of cardiac inflammation and the consequent reparative fibrosis, leading to an important functional loss of the organ over the years. The therapeutic use of the rAdVax vaccine with T. cruzi antigens showed a promising effect in the experimental chronic phase with reversal of cardiac lesions, including fibrosis.
TNF also stands out as an important cytokine involved in the chagasic cardiac dysfunction. Experimental models of chronic infection developed by Lannes-Vieira et al. demonstrated an increase in circulating levels of TNF and nitric oxide, both associated with the severity of cardiac involvement. These models are efficient in the search for new therapeutic compounds focusing on the clinical manifestations observed in the chronic phase of the disease. The efficiency of the combined therapy of benznidazole with the immunoregulatory agent, pentoxifylline, was demonstrated in experimental chronic chagasic cardiomyopathy, with a potential effect on the reduction of fibrosis, myocarditis, parasite load and improvement in electrical changes. These findings support the importance of using combined treatments with direct action on the etiologic agent and that regulate the exacerbated immune response to improve the prognosis of patients with chronic chagasic heart disease.
René Rachou Research Center/Fiocruz
Alexandre Barcelos Morais da Silveira
René Rachou Research Center/Fiocruz
Débora d’Ávila Reis
Department of Morphology, ICB, Federal University of Minas Gerais
Megaesophagus, megacolon, and heart disease are causes of morbidity and mortality in the chronic phase of Chagas’ disease. Our laboratory has been particularly interested in the study of the immunopathology of Chagas’ megaesophagus and megacolon. Epidemiological studies in endemic areas of Brazil have shown that 8-10% of chronic patients have the digestive form of the disease.Patients with the digestive form present a series of symptoms related to organ obstruction. In both megaesophagus and megacolon, the organs exhibit large lumen enlargement and muscle layer hypertrophy. Histological analyzes of Organs affected organs have demonstrated inflammatory lesions of the enteric nervous system (ENS) (Figure 1), associated with a large reduction in the number of neurons. According to Koberle, for the development of megaesophagus, a reduction of about 85% in the number of neurons in the organ is necessary, while colon disease is associated with a neuronal loss of at least 50%.
Although the mechanism of neuronal injury remains unclear, the frequent observation of ganglionitis and periganglionitis in patients with mega points to the participation of immune system cells in this process. The role of the parasite has been investigated, both in inflammatory processes of the acute phase and in lesions of the chronic phase. We demonstrated the presence of parasite kDNA in the esophagus of chronic patients with megaesophagus and megacolon (unpublished data), thus demonstrating a strong association between the development of mega and the persistence of the parasite in the chronic phase. In LSSP-PCR (Low-stringency Single Specific Primer-PCR) studies, we also demonstrated similarity between the kDNA signatures obtained from parasites in the esophagus of two different patients with severe megaesophagus.
More recently, we observed that Trypanosoma cruzi kDNA is also detected in esophageal or colon samples from about 50% of patients without mega, contrary to what had been demonstrated by immunohistochemical studies. These observations prompted us to re-discuss the pathogenesis of mega from another aspect. To understand this pathological process, we must analyze not only the presence of T. cruzi in the organ, but also consider the variability of parasite strains and other factors related to the host, such as genetics, immune response and the denervation process.
In later studies, we observed a strong association between the presence of cells with cytotoxic potential, denervation process and megaesophagus development, and evidence of the occurrence of similar cytotoxicity mechanisms was also evidenced later in the chagasic megacolon. Some individuals without mega also presented denervation and inflammation, but less intense. However, certain patients without mega and without any digestive symptoms showed a reduction in the number of ENS neurons close to the threshold previously established by Koberle. These data prompted us to start a comparative neurochemical study of the ENS in patients with and without megacolon. Knowing that the ENS contains about 10 to 100 million neurons, with a wide variety of neurotransmitters and/or neuropeptides, we hypothesized that, for the development of megacolon, it was necessary to have a selective destruction of certain classes of neurons, the that would affect peristalsis and vascular tone and, consequently, would favor the development of the pathology.In fact, our group was the first to demonstrate that the development of chagasic megacolon seems to be explained not only by the rate of neuronal death, but also by the frequency of destruction of each neuron class. The selective destruction of nNOS+and VIP+ inhibitory motor neurons and the increased frequency of substance P+ neurons seem to favor the development of this pathology (Figure 2). We also demonstrated that the destruction of enteroglial cells in chagasic infection seems to happen early and is not correlated with the development of megacolon and that possibly GFAP+ enteroglial cells participate in the modulation of the inflammatory process and, consequently, in neuronal protection and control. of the development of chagasic megacolon.
The changes suffered by neuroimmune integration have been the subject of research in several pathologies that affect the gastrointestinal tract. In Chagas’ disease, we believe that the study of the enteric nervous system, as well as its association with the inflammatory process, may provide subsidies for the understanding of neuroimmune alterations that may be somehow involved in the development of mega.
The neuropeptides found in the ENS have considerable activity on the immune system. For example, substance P is considered a protein that, in addition to being a neuromediator, has a pro-inflammatory action on immune system cells. It stimulates lymphocyte proliferation, lymphocyte traffic through lymph nodes, and IL-2 production. In addition, substance P acts as one of the activators of Natural Killer (NK) cells and has chemotactic action for mast cells, macrophages, and neutrophils. The VIP neuropeptide inhibits the response of NK cells and T lymphocytes, as well as the production of IL-2 and IL-4 by these cells. On the other hand, VIP stimulates macrophage chemotaxis and IL-5 production by lymphocytes. In this context, it is interesting to highlight our findings of increased frequency of substance P+ neurons in the colon of megacolon patients, which suggests their participation in the maintenance of the inflammatory process of the chronic phase.We emphasize the preliminary data from our laboratory, resulting from immunohistochemical studies for the protein GAP-43, a marker of neuronal regeneration (Figure 3). The preliminary results obtained suggest that this process of neuronal plasticity is occurring in the colon of patients with chronic infection. Based on these data, a new investigative line was opened in the study of the pathology of chagasic megacolon. To understand the development of this pathology, we must assess not only the degree of destruction of the different neuronal classes, but also their regeneration rate.
Another important aspect to be evaluated is the expression of neurotrophins. Neuronal growth factor (NGF) was the first neurotrophin to be described, once its role in the development, differentiation and maintenance of sensory and sympathetic neurons was established. A number of cells produce NGF, among which we can highlight neurons, glial cells, muscle cells, fibroblasts and even some cells of the immune system, such as B lymphocytes and T lymphocytes. In addition to NGF, a series of neurotrophins came to be characterized due to their effects on nervous tissue. Among them, we highlight the Ciliary Neurotrophic Factor (CNTF), the acidic (a-FGF) and basic (b-FGF) Fibroblastic Growth Factors, and the Glial Cell-Derived Neurotrophic Factor (GDNF), among others.
GDNF is a polypeptide originally known for its stimulating action on central nervous system neurons, such as midbrain dopaminergic neurons and motor neurons. GDNF is also responsible for the development and survival of enteric neurons. Substantial trophic effects of GDNF on populations of autonomic neurons, particularly ENS neurons, have been shown to be distinct from those produced by other neurotrophins and neurotrophic factors. As with NGF, neurons, glial cells and muscle cells are the main producers of GDNF.
By unraveling certain aspects related to the establishment and development of Chagas’ megacolon, we believe that we will be contributing not only to the application of new preventive and therapeutic methodologies in Chagas’ disease, but also to a wide spectrum of diseases that afflict the digestive system, such as Chron’s disease, inflammatory bowel disease, irritable bowel syndrome, and Hirschsprung’s disease. Furthermore, we hope that the data resulting from our line of research can serve as a guide for future experimental work on the pathogenetic mechanisms of T. cruzi infection.
Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro
In this module, we will report on the evolution of research on the structure and biological function of the cysteine proteases of Trypanosoma cruzi. Identified as a therapeutic target in the mid-1990s, the major isoform, cruzipain, is now recognized as a virulence factor in T. cruzi. In addition to generating peptides that stimulate the internalization of trypomastigotes by cardiovascular cells, the enzymatic activity of cruzipain is essential for the development and differentiation of intracellular amastigotes. More recent studies indicate that cruzipain generates pro-inflammatory peptides at sites of infection, alerting sentinel cells of the innate immune system to the presence of trypomastigotes in the interstitial tissue. This perception came to light due to the characterization of kininogens (bradykinin precursors) as natural substrates of the major isoform (cruzipain). The narrative of this text will be divided into two parts: in the first, I will describe the background of this research. Next, I will describe more recent findings, demonstrating that the activation of the Kinin System by the T. cruzi has an impact on the immunopathogenesis of experimental Chagas’ disease.
In the mid-1970s, Erney Camargo et al. were the first to describe the presence of proteolytic enzymes in T. cruzi extracts. Shortly afterwards, Rangel et al. partially described the physicochemical properties of an epimastigote thiol protease. At the end of that same decade, there was a convergence between 3 lines of research that until then had been developing in parallel in Buenos Aires, Rio de Janeiro, and San Francisco. In a pioneering work, Cazzulo et al. purified and characterized the enzymatic properties of the main lysosomal cysteine protease of T. cruzi, called “cruzipain”. Shortly thereafter, McKerrow et al. (UCSF) described the partial oligonucleotide sequence of a T. cruzi cysteine protease that displayed the characteristic catalytic triad (Cys 25, His 159, and Asn 175) of papain superfamily enzymes. At the same time, our team demonstrated that cruzipain was identical to GP57/51, a glycoprotein previously defined by our team as a dominant T. cruzi antigen. In addition to GP57/51 having an N-terminal sequence virtually identical to cruzipain, we obtained evidence that it was a thiol protease of the papain family (sensitive to E-64 and cystatin), capable of degrading high molecular weight protein substrates, both at neutral and acidic pH. Using monoclonal antibodies a-GP57/51, we verified that the antigen had an eminently lysosomal localization pattern (reservosomes).
Before describing the knowledge obtained on the structure-function relationships of cruzipain, it is opportune to mention the historical background of this research. In 1983, Mendonça-Previato et al. had isolated and characterized several fractions from aqueous (boiled) extracts of epimastigotes. Among others, the glycoconjugate of 25,000 daltons, called GP25, stood out for its high content of O-linked oligosaccharide chains. When analyzing the antigenicity of GP25, we found that the glycoprotein could be used as a tool in the serodiagnosis of Chagas’ Disease. Taking advantage of the availability of high amounts of GP25, we isolated human IgG antibodies through affinity chromatography. The analysis of the GP25 expression profile revealed the presence of epitopes in all stages of parasite morphogenesis. Epimastigotes exhibited a strong density of epitopes on the cell surface, but the surface reaction of trypomastigotes (blood forms, BFT) was only mild. Despite the slight reaction of human IgG antibodies a-GP25 with the surface of BFT, it was surprising to find that these antibodies were able to significantly reduce the infection of human smooth muscle cells (primary culture). Interestingly, tests carried out in parallel with fibroblasts (primary culture) obtained from the same donors showed that the infectivity of the parasite had not been impaired by the action of these antibodies. Evidently, in 1982, it was not possible to explain the reasons for the differential effect of human IgG antibodies a-GP25 in these two models of cellular interaction. Furthermore, the molecular characterization of GP25 was proving difficult, because the N-terminal end of the (glyco)protein seemed to be blocked, making its sequencing unfeasible. Furthermore, the isoelectric pattern of GP25 was quite complex, suggesting the existence of structural microheterogeneity. These difficulties were partly overcome by obtaining monoclonal antibodies a-GP57/51. Analysis of the immunoprecipitation pattern of antigens metabolically labeled with [35 S]-methionine revealed an important finding: epimastigote lysates that had been previously treated with a protease inhibitor cocktail showed only two characteristic bands of 57/51,000 daltons, referred to as thereafter as the GP57/51 antigen. The premise that GP57/51 was sensitive to proteolysis was confirmed when protease inhibitors were omitted from the preparation of cell lysates: under these conditions, the monoclonal antibody precipitated antigenic fragments of MW~25 KDa. After proving that monoclonal or polyclonal a-GP25 antibodies reacted strongly with FPLC-purified GP57/51, we concluded that the dominant epitopes of GP25 were shared with GP57/51. Based on this set of observations, we deduced that GP57/51 (cruzipain) was the primary biosynthetic product produced by epimastigotes. As a corollary, we deduced that GP25 was a stable product generated by the proteolysis of cruzipain.
It was up to Cazzulo’s team to determine the sequence (130 amino acids) of the C-terminal domain of cruzipain. Containing several predictive sites of N/O-glycosylation, this C-terminal extension joins the catalytic (central) domain through a (folding-like) sequence rich in threonine and prolines. As predicted, the folding flanks contained sequences sensitive to cleavage by autoproteolytic mechanisms.
Shortly thereafter, complete genomic DNA clone sequencing showed that cruzipain is a single-chain polypeptide synthesized as a pre-pro-enzyme. The PRE region is composed of a hydrophobic peptide capable of directing the molecule to the endoplasmic reticulum. As with other members of the papain superfamily, the PRO-peptide segment is critical to the precursor protein folding process. In addition, the PRO segment binds to the catalytic domain of several enzymes of the papain family, blocking the access of substrates to its interior of the catalytic cleft. As described for other C1 family enzymes, cruzipain was predicted to have a “V”-shaped cleft stabilized by the interaction of 2 adjacent subdomains.
Initially, the task of obtaining enzymatically active forms of cruzipain from inclusion bodies was not successful, which delayed the analysis of its three-dimensional structure. Despite this, obtaining the inactive recombinant protein allowed the resumption of immunological studies. After obtaining continuous lines of CD4 lymphocytes isolated from carriers of Chagas’ disease, we evaluated the antigenic specificity of these lines using a panel of overlapping synthetic peptides, covering the catalytic and C-terminal domains. These studies revealed the presence of immunodominant T epitopes in the core domain. Years later, the fine specificity of intracardiac CD8+ T lymphocyte lines from patients with Chagas’ disease was characterized by the team led by Cunha-Neto. Results from patient studies indicated that T epitopes are concentrated in the central (catalytic) domain (18,20), whereas epitopes recognized by human IgG antibodies are concentrated in the C-terminal domain (i.e., on GP25). Years after, studies performed in experimental vaccination models indicated that cruzipain has an immunoprotective effect. More recently, researchers from the University of Cordoba have shown that the genetic background of mice influences the TH pattern developed by animals immunized with enzymatically inactive forms of cruzipain.
While the UCSF team was making efforts to obtain the active form of recombinant cruzipain, our group continued to study the substrate specificity of natural cruzipain isolated from epimastigotes (Dm28c strain). As proposed by the team from Cazzulo, in Argentina, the enzymatic assays performed by Ana Paula Lima and collaborators confirmed that cruzipain was an enzyme related to L cathepsin. Reflecting the conformational particularities of this enzyme, we recorded that its activity (L cathepsin-like) enzyme was subject to inhibition by excess substrate. Interestingly, this inhibition occurs at room temperature, being reversed at 37oC. Based on these observations, we suggest that cruzipain was able to self-regulate its enzymatic activity during the development of T. cruzi in triatomines. In another unprecedented observation, the analysis of pH profiles obtained with different synthetic substrates indicated that cruzipain also behaved as a B cathepsin. This effect was credited to the presence of a glutamic acid at position 205 in the S2 subsite, involved in the interaction with Arg at the P2 position of peptide substrates. Years later, the analysis of the crystal structure of the catalytic domain of cruzipain (conventionally called “cruzaine” by the American team) would confirm our thesis that cruzipain has mixed specificity (L/B cathepsin)
The first indications that the expression of GP57/51 (cruzipain) was regulated during metacyclogenesis (Dm28c) were obtained in studies performed in collaboration with the team led by Samuel Goldenberg, from Fiocruz. As in parasitized mammalian cells (amastigotes>trypomastigotes), we noticed that the expression of the major isoform was much higher in replicative forms (epimastigotes>amastigotes) than in metacyclic trypomastigotes. The isoelectric profiles showed heterogeneity, but at that time it was not possible to conclusively determine whether the observed complexity was just a result of post-translational modifications (e.g. differences in glycosylation pattern) and/or the existence of polymorphic genes in the Dm28c clone. Shortly thereafter, it was shown that cruzipain was a member of a family of genes arranged in a repetitive arrangement. Motivated by these findings, Ana Paula Lima sequenced several cruzipain cDNAs derived from the T. cruziDm28c cDNA library. Analysis of these sequences revealed that some sequences had point substitutions of amino acid. Based on molecular modeling, we deduced that substrate interaction pockets could be altered in some isoforms. Years later, the analysis of the substrate specificity of recombinant cruzipain 2 (catalytic domain) expressed in S. cerevisae would prove that this minority isoform has substrate specificity and catalytic properties very different from the majority isoform. It should be noted that in later works, we demonstrated that there are important differences in the expression profile of these two isoforms, suggesting that different members of the cruzipain family can be differentially regulated during the different stages of the protozoan life cycle. The importance of post-translational regulation of cruzipain isoforms became even more obvious when Ana Carolina Monteiro et al. characterized a potent endogenous inhibitor of cysteine proteases, the “chagasin” protein, named after Prof. Carlos Chagas Filho. Subsequently identified in other protozoa and even in bacteria (but absent in mammals), the chagasin family proteins are encoded by gene sequences completely different from the cystatins (i.e., inhibitors of cysteine proteases expressed in mammals). NMR studies and analysis of chagasin crystals by X-ray diffraction revealed that chagasin has a peculiar three-dimensional structure, containing regions homologous to the CDRs of immunoglobulins in their binding sites for cysteine proteases.
As highlighted by Monteiro et al., the expression of chagasin decreases in the stages of parasite replication (epimastigotes/amastigotes), coinciding with increased expression of cruzipain. This inversion in the stoichiometric relationship between cruzipain/chagasin suggests that the nutritional demand associated with cell division requires maximization of lysosomal protein catabolism, mediated in part by cruzipain, by minority isoforms, and by B cathepsin, previously described by Santana et al. In contrast to epimastigotes/amastigotes, trypomastigotes have relatively high levels of chagasin, again inversely related to low levels of cruzipain. These observations seemed to suggest that extracellular functions of cruzipain (for example, related to cell invasion) were modulated by chagasin. This working hypothesis was recently tested using genetically modified parasites. The results showed that the infectivity of trypomastigotes decreases when the expression of chagasin increases, due to the reduction of the extracellular enzymatic activity of cruzipain. In summary, the set of these studies indicates that the genetic polymorphism generated a diversified repertoire of cruzipain isoforms, some of which have peculiar enzymatic functions. Differently subject to control by chagasin, the cruzipain isoforms can exert specialized functions, possibly favoring the adaptation of T. cruzi both in the triatomine and in the vertebrate host. It has not yet been determined whether the repertoire of cruzipain isoforms present in the phylogenetic lineages T. cruzi I and II was preserved or altered during evolution.
In the mid-1990s, the development of selective inhibitors for cruzipain would become a valuable achievement in basic research in Chagas’ Disease. These tools were indispensable for studying the biological role of cruzipain, in view of the technical difficulties encountered in knocking out the multiple genes that encode these isoenzymes. In 1992, we published the first studies indicating that cruzipain could be an important therapeutic target. We had the opportunity to address this issue using the primary cardiomyocyte cultures that Maria Nazareth Meirelles had standardized at Fiocruz. Aiming to develop new inhibitors of L cathepsin-like enzymes, Luiz Juliano (UNIFESP) synthesized a panel of peptide derivatives with the diazomethylketone group (reactive for active-site cysteine 205) at the C-terminal end. Before testing them in the cell culture system, we performed enzymatic assays with the purified cruzipain to determine the inhibition constants of each compound. These studies indicated that cruzipain was efficiently inactivated by compounds that had bulky aromatic residues at the P2 position (for example, Z-(SBz)Cys-Phe-CHN2 (31). When added to cultures of pre-infected cardiomyocytes, compounds that had this characteristic showed a strong anti-parasitocidal effect, especially Z-(SBz)Cys-Phe-CHN2 (31). To verify if cruzipain had actually been targeted by the aforementioned inhibitors, we treated the parasitized cells with radio-iodinated peptide inhibitors (substituted by tyrosine), obtained cell lysates, and then treated them with a-GP25 antibodies. Analysis of the immunoprecipitated products revealed the presence of radiolabeled inhibitors in the cruzipain immune complexes, thus confirming that the enzyme was targeted in the cultures of parasitized cardiomyocytes (31). Complementing these studies, we demonstrated that the addition of Z-(SBz)Cys-Phe-CHN2 in cardiomyocyte cultures exposed to extracellular trypomastigotes significantly inhibited cell invasion. Based on these data, we suggest that cruzipain (and/or minority isoforms) were virulence factors of T. cruzi. Soon after the publication of these data, Harth et al. showed that peptide inhibitors containing a fluoromethylketone reactive group inhibit the intercellular transmission of the parasites in cell culture. Unfortunately, the cruzipain inhibitors used at that time could not be used in vivo because of toxicity.
As mentioned earlier, the elucidation of the crystallographic structure of the catalytic domain of cruzain (complexed with irreversible inhibitors) paved the way for the synthesis of more effective and low-toxic drugs. Using irreversible inhibitors containing the vinylsulfone group at the C-terminal, Engel et al. were able to cure mice infected with T. cruzi. More recently, pre-clinical studies (in immunodeficient canines and mice) performed with the latest generation of cruzipain inhibitors have proved the therapeutic potential of these drugs.
Despite the progress achieved in the development of drugs capable of inactivating cruzipain, the mechanism of action of these enzymes is still not well understood. The first clue about the nature of the natural substrates of cruzipain emerged when Elaine Del Nery, Luiz Juliano et al. examined in more detail the substrate specificity of the purified enzyme. By testing the protease against a broad panel of synthetic peptides (containing intramolecular fluorescence quenchers), Del Nery et al. verified that cruzipain hydrolyzes peptides that present the bradykinin flanking sequences in kininogen molecules. These observations seeded the idea that the parasite was capable of releasing kinins, similarly to what happens with tissue kallikrein. As I will report later, this hypothesis was later proven in several experimental models.
Before discussing the mechanism of activation of the kinin system by T. cruzi, it is worth making considerations about the multi-modular structure of kininogens. HMW-K (626 amino acids) is organized into 6 domains (called D1-D6), described in table 1, with only the first 4 shared with LMW-K.
Table 1. Modular structure of HMW-K
Kinins constitute a restricted group of peptides of 9-11 amino acids structurally related to the bradykinin (BK) nonapeptide. The first group of kinins includes only the primary products released proteolytically from kininogens: BK (nonapeptide) or lysyl-BK (LBK, also called kallidin). BK/LBK act as paracrine hormones, stimulating transmembrane receptors coupled to heterotrimeric G protein (B2 subtype) of bradykinin. Among several important biological functions, activation of the constitutive B2R receptor induces vasodilation, increased vascular permeability and pain sensitivity, smooth muscle contraction, and, as described later, dendritic cell maturation. The distant effect of BK/LBK on B2R is controlled by enzymes that degrade them, for example by the angiotensin-converting enzyme (ACE). While B2R is constitutively expressed on the cell surface (endothelial cells, smooth muscle, epithelial cells, fibroblasts, neuronal cells, and dendritic cells), the B1 receptor subtype has low expression in normal tissues. However, cellular expression of B1R increases due to activation of MAP kinases and nuclear transcription factor kappa B (NF-kB), in response to injury, infection or inflammation. It is important to highlight that the pharmacological specificity of B1R differs from that of B2R: B1R agonists ([des-Arg9]-BK and [des-Arg]-LBK) are metabolites generated by the proteolytic removal of the C-terminal Arg of BK or LBK, by the action of kininase I (M/N carboxypeptidase). In chronic inflammatory states, activation of B1R induces recruitment of circulating leukocytes to tissues, contributing to tissue fibrosis and angiogenesis.
At first, the notion that cruzipain was a “kininogenase” seemed paradoxical to us, because Kininogens (soluble) are potent inhibitors of cysteine proteases, due to the presence of (cystatin-like) D2/D3 domains. In fact, we had already demonstrated that cruzipain forms molecular complexes with HMW-K/LMW-K, resulting in the blockage of the enzymatic activity of cruzipain. There was yet another reason why we were so reticent at the time: incubation of soluble HMW-K with cruzipain resulted in a slow release of LBK, suggesting that kininogenase activity was inefficient. Faced with this question, we decided to verify whether HMW-K molecules adsorbed on the cell surface were capable of inhibiting cruzipain with the same efficiency as the soluble forms of kininogens. This hypothesis was supported by published findings indicating that the binding sites of HMW-K to the endothelial cell are present in the D3 domain, i.e., they could impair the interactions of the cystatin-like structure with the active site of cruzipain. In addition, work published by other groups has demonstrated that histidine-rich (positively charged) domain (D5) of HMW-K binds with negatively charged groups of sulfated proteoglycans arranged on the extracellular matrix and/or cell surface. Consistent with this premise, we found that the catalytic efficiency of cruzipain increased dramatically when the enzyme was incubated with heparan sulfate (HS). Furthermore, the interaction with HS restricted the effectiveness of the inhibitory effect of HMW-K on cruzipain peptidase activity. To verify whether HMW-K available on the surface of cells was able to serve as a substrate for cruzipain, we used human umbilical cord cells (HUVECs) or CHO-B2R cells (transfected) as indicators of kinin generation. [Ca2+]i influx measurements proved that cruzipain was able to stimulate B2R receptors. The enzyme did not induce a significant influx of [Ca2+]i in CHO-mock (controls). Furthermore, the CHO-B2R response was blocked by E-64. Complementing these studies, we repeated this analysis using cell-cultured trypomastigotes (TCT) of Dm28c strain. The results showed that the parasites were able to generate agonists (BK/LBK) of the B2R subtype bradykinin receptor through the proteolytic activity of cruzipain. These results support the hypothesis that cell surface-associated HMW-K (for example, CHO-B2R or HUVECs) lose the ability to inhibit cruzipain, becoming sensitive to the proteolytic attack of this kininogenase. The identification of angiotensin-converting enzyme (ACE) as a (negative) regulatory factor of cellular activation mediated by the cruzipain>bradykinin>B2R axis was another important observation made in this work. Although there is no evidence that these events promote the infection of cardiovascular cells in vivo, in vitro studies show that the parasite can use cruzipain to invade cells that express subtype B2R or B1R bradykinin receptors (induced during inflammation). It is worth mentioning that only the hydrophobic cysteine protease inhibitors were able to inhibit the invasion of HUVECs or CHO-B2R, but not of CHO-mock. These results suggested that kininogens (cruzipain substrate) are hydrolyzed in the contact regions between the plasma membranes of the parasite and the target cell. Ongoing studies aim to clarify whether the activity of cruzipain (presumably secreted in the flagellar pocket region) is potentiated by interactions with other cell surface elements.
Continuing the studies on the role of cruzipain in the pathophysiology of experimental Chagas’ disease, we verified whether TCTs were able to activate the kinin system in vivo. Using intravital microscopy, we demonstrated that trypomastigotes applied topically to hamster cheek pouch tissue induce an increase in capillary permeability (extra leakage of dextran blue particles) through the cruzipain>BK/LBK/B2R axis. Upon inspecting the postcapillary veins, we observed that TCTs induce leukocyte margination within a few minutes, preceding the onset of plasma leakage. It is noteworthy that the topical application of low concentrations of cruzipain did not induce significant plasma extravasation. However, the addition of an exogenous source of HMW-K together with cruzipain produced a strong increase in capillary permeability via the cruzipain/B2R-dependent pathway. Based on these observations, we decided to explore the possibility that the effect of cruzipain is conditioned by the influx of plasma (HMW-K/LMW-K source) into the extravascular tissue. In other words, we deduced that TCTs induced, albeit discreetly, the influx of plasma proteins (containing kininogens) through another activation pathway, hitherto undefined. This hypothesis seemed plausible to us because the injection of epimastigotes (which express abundant amounts of cruzipain) at a dose equal to the trypomastigote inoculum did not induce significant plasma extravasation in the hamster cheek tissue, nor did it induce paw edema in mice, even after treatment of animals with captopril (ACE inhibitor). At that time, Ricardo Gazzinelli, Igor Almeida et al. had already demonstrated that TCT expressed high levels of a pro-inflammatory lipid anchor (tGPI-mucin), characterized as a TLR2 ligand. It should be noted that epimastigotes express low amounts of this ligand. In fact, the explanation of the mechanism of action of cruzipain emerged when we examined the contribution of TLR ligands in the activation mechanism of kinins (cruzipain>BK/LBK>B2R axis), using the TCT-induced edematogenic inflammatory response (Dm28c) in wild-type and TLR2, TLR4 and B2R-deficient mice as a biological indicator. In addition to demonstrating that the activation of neutrophils via TLR2 promotes the accumulation of kininogens in the extravascular tissue, we show that these events influence (i) the production of IL-12 by dendritic cells recruited to the draining lymph nodes and (ii) the anti-T. cruzi TH response profile. Based on these results, we conclude that inflammation induced by the cooperation between TLR2/B2R stimulates TH1 responses in the subcutaneous infection model. The works that demonstrate the importance of activation of dendritic cells by bradykinin (previously defined as an endogenous signal of maturation) will be described in the next chapter. For now, interested readers can benefit from reading original articles recently published by our team.