Download Is there a pathogenic role of autoimmune responses in Chagas

Revisión
Inmunología
Vol. 23 / Núm 2/ Abril-Junio 2004: 185-199
Is there a pathogenic role of autoimmune
responses in Chagas’ disease?
N. Gironès, H. Cuervo, M. Fresno
Centro de Biología Molecular, CSIC-UAM, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain
¿EXISTE ALGÚN PAPEL PATOGÉNICO PARA LA AUTOIMMUNIDAD EN LA ENFERMEDAD DE CHAGAS?
RESUMEN
Trypanosoma cruzi es el agente causal de la enfermedad de
Chagas, un paradigma de enfermedad autoimmune inducida por
infección. La escasez de parásitos en la fase crónica de la enfermedad contrasta con la severa patología cardíaca observada en
aproximadamente un 30% de los pacientes crónicos, y sugiere un
papel para la autoinmunidad en el orígen de la patología. Dependiendo de la respuesta inmunitaria contra el parásito, se han descrito mecanismos antígeno específicos y antígeno no-específicos
que pueden activar células T y B y causar autoinmunidad. Entre
los primeros, el mimetismo molecular (similitud de secuencias
entre antígenos del agente infeccioso y del hospedador) se considera el mecanismo más importante que puede conducir a autoinmunidad y patología en la fase crónica de esta enfermedad. Con
el uso de técnicas más sensibles, el parásito se detecta cada vez
más facilmente en el hospedador infectado, el cual puede padecer patología bien directamente o a través de la respuesta inflamatoria específica contra el parásito. Por ello, el tema de la autoinmunidad versus persistencia del parásito como causa de la patología en la enfermedad de Chagas es extensamente debatido entre
los investigadores del área. Nuevos argumentos a favor y en contra de cada hipótesis han sido publicados recientemente. En el
presente trabajo pasamos revista a ambas hipótesis de forma crítica y desde perspectivas clínicas, patológicas e inmunológicas.
PALABRAS CLAVE: Trypanosoma cruzi / Enfermedad de Chagas
/ Autoinmunidad / Mimetismo molecular.
ABSTRACT
Trypanosoma cruzi is the pathogenic agent responsible for the
Chagas´ disease, a paradigm of infection–induced autoimmune
disease. The scarcity of parasites in the chronic phase of the disease contrasts with the severe cardiac pathology observed in approximately 30% of chronic patients, and suggests a role for autoimmunity as the origin of the pathology. Depending on the antiparasite immune response, antigen-specific and antigen-non-specific mechanisms have been described by which T. cruzi infection
might activate T and B cells leading to autoimmunity. Among the
first ones, molecular mimicry (sequence similarity between infectious agents and self-proteins) has been claimed as the most important mechanism leading to autoimmunity and pathology in the
chronic phase of the disease. The use of more sensitive techniques
has led to the easy detection of the parasite in the infected host,
who can undergo pathology either directly or through parasitespecific mediated inflammatory responses. Thus, the issue of
autoimmunity versus parasite persistence as the cause of Chagas’
disease pathology is hotly debated among many researchers of
the field. Lately, there have been numerous reports offering arguments in favour of one or another hypothesis. We critically review
here both hypotheses from clinical, pathological and immunological perspectives.
KEY WORDS: Trypanosoma cruzi / Chagas’ disease / Autoimmunity / Molecular mimicry.
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IS THERE A PATHOGENIC ROLE OF AUTOIMMUNE RESPONSES IN CHAGAS’ DISEASE?
TABLE I. Mechanisms for activation of T and B cells in autoimmune diseases
VOL. 23 NUM. 2/ 2004
TABLE II. Criteria required for demonstration of the involvement of molecular mimicry in an autoimmune disease
a. Microbial antigen specific:
• Molecular mimicry between parasite and host antigens triggers autoimmunity
1. Association of the disease with a particular microorganism.
b. Microbial antigen non-specific:
• Release of autoantigen(s) during infection
• Bystander activation
• Cryptic epitopes
• Superantigens
3. T or B cell populations against that epitope should be expanded in the infection.
ROLE OF INFECTIOUS DISEASES IN AUTOIMMUNITY
The idea that infection plays an important role in initiating
autoimmune disease dates back more than a century and
is one of the most enduring paradigms in the annals of
autoimmunity(1-3). To understand how microbial infections
might cause autoimmune diseases it must be taken into
account that immunocompetent individuals harbour
potentially autoreactive T and B lymphocytes, that are
normally tolerant to self antigens(4). These cells remain
innocuous unless somehow activated. The infection is
thought to be the trigger of that activation. For example, in
some animal models such as experimental autoimmune
encephalomyelitis (EAE) or the non-obese diabetic (NOD)
mouse model for type I diabetes, only activated but not
resting T cells can transfer the disease(5,6).
Two main classes of mechanisms have been
described by which infectious agents might activate T
and B cells and lead to autoimmunity: antigen-specific and
antigen-non-specific (Table I). In the microbial antigenspecific mechanism the sequence similarity between infectious
agents and self-proteins (molecular mimicry or epitope
mimicry) might trigger the autoimmune response(1-3,7,8).
Antigenic determinants on the infecting microorganism’s
proteins are structurally similar to one or various determinants
on the host proteins, but are different enough to be recognised
as foreign by the immune system of the host. Many reported
evidences indicate that molecular mimicry is commonplace
and many sequential and structural determinants of infectious
agents have been shown to stimulate crossreactivity with
epitopes on host tissues(1-3,7,8). Autoreactive B and/or T cells
in response to foreign antigens originated by molecular
mimicry can arise from a T/B-cell cooperation mechanism,
but direct experimental evidence is still scarce(1,3). Moreover,
there is, as yet, no absolute formal proof that molecular
mimicry is the initiating cause of human autoimmune disease
and responsible for the pathology, as remarked recently(9,10).
Probably, Chagas’ disease is close to that paradigm. To
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2. Identification of the culprit microorganism epitope that elicits
the cross-reactive response.
4. Elimination of the cross-reactive epitope from the microorganism should result in non-pathogenic infection.
5. Autoreactive T cells should be able to transfer the disease.
prove the involvement of epitope mimicry in a disease of
suspected autoimmune aetiology five criteria need to be
confirmed experimentally(9,11) (Table II).
The microbial antigen non-specific mechanism of
autoimmunity has several variants. The common characteristic
is that no particular microbial determinant is implicated,
although the infection may be the initial event that triggers
the autoimmune reaction. For example, infection might
cause destruction of host cells, which results in the release
of large quantities of normally sequestered proteins. Those
cryptic epitopes found in intracellular proteins are not
normally presented in the context of class I Major
Histocompatibility Complex (MHC) and are, therefore,
not normally encountered by host lymphocytes. These
could then be captured by dendritic cells that trigger naïve
T cells present at the invasion site or at the T cell areas of
the draining lymphoid organs, leading to activation of
autoreactive cells but not of cells against the infecting
microorganism.
Cryptic epitopes may also initiate and maintain
autoimmunity through various non-mutually exclusive
mechanisms (12). Cryptic epitopes can be presented by
non-professional antigen presenting cells (APC) such as B
cells, and induce T cell activation. Autoreactive B cells initiate
autoimmunity in the absence of T cells specific for the selfantigen. Alternatively, autoreactive B cells may take up a
foreign antigen that cross-reacts with a self-antigen at the
B cell level, but contains different T cell epitopes. Finally,
activated B cells that efficiently take up and present selfantigen may prime autoreactive T cells. All these mechanisms
may result in a self-sustained autoimmune response.
Alternatively, or in addition, microbial infection may
result in bystander activation, which may take place in the
setting of a proinflammatory milieu. Thus, microbial infection
induces the release of proinflammatory cytokines such as
tumour necrosis factor-α (TNF-α) and chemokines, that
could be able to activate autoreactive T cells by lowering
INMUNOLOGÍA
N. GIRONÈS, H. CUERVO, M. FRESNO
Figure 1. Infective cycle of T. cruzi.
Transmission of T. cruzi to humans occurs
when infective metacyclic trypomastigote
forms of the parasite (A1), present in the
faeces released by the bug while it takes a
blood meal penetrate in the bloodstream.
They infect a wide variety of phagocytic
and non-phagocytic cells (A2) of the host.
Once inside the cells, the metacyclic forms
escape from endocytic vacuoles to the
cytoplasm where they transform into
amastigotes and multiply intracellularly
(A3). At some point, the amastigotes break
off from the cell (A4), differentiate into nonreplicative flagellated blood trypomastigotes
that in turn penetrate and infect either
adjacent or distant susceptible cells and
tissues of the body (A5a). Amastigotes can
also directly infect phagocytic cells (A5b).
Muscle cells, including those of the heart,
are amongst the most heavily infected. New
triatomine bugs may take up circulating
triypomastigotes during a blood meal (A6).
Inside the vector’s intestine, ingested blood
trypomastigotes differentiate into replicative
epimastigotes, which as they move to the
mid and lower gut they transform into nonreplicative but infective metacyclic
trypomastigotes.
the threshold of activation(13,14). These T cells may then
proliferate in response to self-antigen presented on host
APC. Inflammation could also alter the pattern of lymphocyte
migration and activate APC’s, rendering them more effective
as APC by enhancing antigen uptake and processing, as
well as the cell surface expression of MHC or costimulatory
molecules.
Finally, infection might provoke polyclonal lymphocyte
activation via either a mitogen or a superantigen effect(15).
CHAGAS’ DISEASE
General aspects
Chagas’ disease is a debilitating multisystemic disorder(16)
that affects several million people (approximately 18 million
individuals are infected with Trypanosoma cruzi, with 120
million at risk) in Central and South America(17-19) and is
considered a paradigm of infection-mediated autoimmune
disease. It is caused by the flagellated protozoan parasite
T. cruzi, which has a complex life cycle involving several
stages in both vertebrates and insect vector. T. cruzi has
three different morphologies: 1) epimastigote, which
replicates in the blood-sucking triatomine insect vector; 2)
trypomastigote, which infects cells of vertebrate hosts; and
3) amastigote, which replicates intracellularly in cells of
the host(17,20). Approximately 30% of infected people develop
symptoms of the disease in their lifetime, which include
cardiomyopathy, neuropathies, and dilatation of colon
or oesophagus(17,18).
Transmission of T. cruzi to humans occurs when the bug
takes a blood meal and faeces containing infective metacyclic
trypomastigote forms of the parasite are released. The later
penetrate in the bloodstream and infect a wide variety of
phagocytic and non-phagocytic cells of the host. Once inside
the cells, the metacyclic forms escape from endocytic vacuoles
to the cytoplasm where they transform into amastigotes,
which multiply intracellularly (Fig. 1).
Individuals residing in rural areas of Latin America are
at highest risk of infection, since the bugs live in these
dwellings and feed on the inhabitants at night. Treatment
with benznidazol or nifurtimox is effective during the acute
phase of infection, but no treatment exists for the chronic
phase(17,18). To date an effective vaccine is still needed, although
many groups are working on both treatment and vaccination.
The World Health Organization has conducted several
programs for the elimination of the insect vector, with great
results on the incidence of new infections(19). However, silvatic
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IS THERE A PATHOGENIC ROLE OF AUTOIMMUNE RESPONSES IN CHAGAS’ DISEASE?
areas are not affected by these programs and constitute
the reservoir for the parasite. Transfusion with infected blood
and congenital transmission also account for some new
infections. Therefore, transfusion-acquired Chagas’ disease
is becoming a significant health problem in countries
other than Central and South America, especially those
having large numbers of immigrants from that region(21,22).
Immune response
Cytokines play a key role in regulating both immune
response and parasite replication in infected hosts (23).
Macrophages, which can be infected by T. cruzi, also play
a crucial role in the elimination of this parasite. Activation
of monocytes by cytokines released by Th1 cells seems to
be a key process in controlling infection in vitro as well as
in vivo. Thus, IL-12 produced by macrophages in response
to infection mediates resistance to T. cruzi(24). TNF-α and
IFN-γ have been identified as the most important cytokines
involved in the killing of intracellular T. cruzi through a
NO-mediated L-arginine-dependent killing mechanism(25,26).
This was corroborated in vivo, since anti IFN-γ monoclonal
antibody administration results in a drastic increase in
parasitaemia and mortality(27,28). TNF-R1-FcIgG3 transgenic
mice are also more susceptible to T. cruzi infection clearly
indicating a protective role for TNF-α(29). Accordingly, T.
cruzi infection of inducible NO synthase deficient mice
results in an increased parasitaemia(30).
On the other hand, several alterations of the immune
response have been described in this disease. Thus, the
acute infection by T. cruzi is associated with severe
immunosuppression, measured as the loss of proliferative
cell responses to mitogens and antigens, which is thought
to facilitate dissemination and establishment of the parasite
in host tissues. Various cell types have been ascribed to act
as suppressor cells and therefore mediate immunosuppression.
Some reports have pointed out to T cells, including γ/δ T
cells(31) as well as to adherent cells(32) or immature myeloid
cells(30). For example, the depletion of adherent cells partially
restored T cell proliferation of spleen cells(33,34). Besides,
inhibition of IL-2 synthesis(33) and reduced cell surface
expression of IL-2R by splenocyte cultures of infected mice(35),
as well as in human peripheral blood cells(36), has been
accounted to explain this unresponsiveness. By contrast,
elevated levels of IFN-γ and TNF-α are produced by infected
and activated spleen cell cultures(30,37,38). In addition, infection
also alters the shaping of the central and peripheral T-cell
repertoire(39).
Apoptosis of CD4+ T cells has been also described in acute
T. cruzi infection(31), and T cells obtained from infected mice
have an enhanced T cell receptor (TCR) activation-induced
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cell apoptosis that may explain this unresponsiveness(40).
Increased NO secretion(41) has been accounted as one mechanism
responsible for immunosuppression and recently, CD11+Gr1+
myeloid cells have been identified as responsible for this NOmediated immunosuppression(30). Other soluble substances,
including suppressive cytokines such as transforming growth
factor (TGF)-β, IL-4 or IL-10, and prostaglandins, released
upon contact with parasite-derived antigens, are also thought
to be involved(42-44). Finally, elevated levels of IFN-γ and TNFα are produced by spleen cell cultures from infected mice(37),
which in turn induce high levels of NO(30).
On the other hand, T. cruzi has evolved sophisticated
systems to evade the immune system being this parasite
probably one of the best examples of adaptation to the
host(45). The entry and replication of T. cruzi inside macrophages
is required for its dissemination in the host(46,47). T. cruzi has
the capacity to evade the protective immune response
produced by macrophages by escaping from the
phagolysosome to the cytoplasm(20,48), and by altering the
profile of secreted cytokines, such as TNF or IL-12(49).
PATHOGENESIS OF CHAGAS’ DISEASE
Pathological features
Two phases, acute and chronic, can be differentiated in
Chagas´ disease(17,18,50). In the acute phase, few weeks after
infection, a local inflammatory lesion (chagoma or Romaña’s
sign) appears at the site of infection where the metacyclic
trypomastigotes infect and undergo their first rounds of
multiplication. After parasite dissemination through the
body, circulating blood trypomastigotes are easily observed
in blood (parasitaemia) and a small number of patients
develop symptoms of cardiac insufficiency, reflecting an
underlying severe myocarditis. This leads in some cases to
heart failure, which is responsible for the few deaths seen
in acute Chagas’ disease(51,52). Meningoencephalitis may also
occur, especially in some immunosuppressed patients(53).
However, the acute phase mostly remains undiagnosed
without clinically severe symptoms. On the contrary, the
most severe pathology and common manifestations of
the disease develop many years (10 to 30) after the initial
infection with T. cruzi in the so called chronic phase, although
only in around 30-40% of the infected people(17,18,50). During
the chronic phase circulating parasites cannot be observed
by inspection of blood but progressive tissue damage occurs
in the esophagus, colon and heart(17,18).
In the chronic phase, the heart is the most commonly
affected organ. Cardiomyopathy frequently develops, being
congestive heart failure a common cause of death in these
INMUNOLOGÍA
patients. Chronic chagasic cardiopathy (CCC) is thus the
most devastating manifestation of Chagas’ disease. However,
despite affecting about a third of the infected people, the
pathogenesis of CCC is still poorly understood. CCC may
be considered as a progressive disease, in which myocardial
inflammation and fibrosis plays a pivotal role(54-56). Moreover,
higher percentages of severe myocarditis, fibrosis and
myocardial hypertrophy are found in CCC patients with
heart failure compared to patients in indeterminate and
cardiac arrhythmia. Examination of the heart of CCC patients
who have died of heart failure shows biventricular enlargement
with occasional apical aneurysms. Individuals with CCC
often develop mural thrombi, which may cause cerebrovascular
accidents. Histological examination of the heart reveals
diffuse interstitial fibrosis, lymphoid infiltration and damaged
myocytes, all occurring in the apparent absence of parasites.
Fibrosis and chronic inflammation are also detected in
the conduction system of the heart, which may account for
the high incidence of arrhythmias. Megaoesophagus and/or
megacolon may also develop in chronic chagasic patients
which, in the most severe form, can cause life-threatening
malnutrition and intractable constipation.
Mechanisms of pathogenesis
After several decades of research, the aetiology of Chagas’
heart disease, both in humans and in experimental infection
models, is not precisely understood yet. The acute and
chronic phases of the disease share some similar pathological
findings (see above), but their pathogenesis may differ. Up
to date, many pathogenic mechanisms have been described
to explain how cardiac pathology develops. They can be
mediated directly by the parasite or caused by an
inflammatory/immune/autoimmune mechanism or by a
combination of them. Those mechanisms are summarised
below:
a) Primary neuronal damage and denervation of the
parasympathetic autonomous system in the heart. This may
lead to the development of chronic phase lesions and was
one of the first pathogenic mechanisms described during
the acute phase(57,58). However, subsequent studies show
only slight neuronal damage in the heart, suggesting that
neuronal lesions are an epiphenomenon, secondary to
inflammation and fibrosis(59-61).
b) Secreted T. cruzi product (s). They can be toxic for
host cells and tissues(62).
c) T. cruzi-induced damage of cardiomyocytes. This is
due to the cytopathic effect of the intracellular infection
with amastigotes. It is an obvious mechanism, which may
have some relevance only in the acute phase and in heavily
parasitised or immunosuppressed patients.
N. GIRONÈS, H. CUERVO, M. FRESNO
d) Parasite-induced microvascular changes. They may
lead to cardiac hypoperfusion, myocyte degeneration and
finally to chronic inflammation(63-65).
e) Persisting T. cruzi antigens. They may act as a trigger
for specific T-cell mediated responses such as delayed-type
hypersensitivity (DTH) or cytotoxic CD8+ cells that lead to
damage of infected cells or bystander cells in the host
tissues(66-68). The latter may take place in both the acute and
the chronic phases. In this regard, a role for cell adhesion
molecules and integrin receptors, extracellular matrix (ECM)
components, matrix metalloproteinases and chemokines
has been proposed in the differential recruitment and
migration of T. cruzi-elicited CD8+ and inflammatory cells
into the heart and other susceptible tissues of the host(69,70).
ECM components may absorb parasite antigens and cytokines
that could contribute to the establishment and perpetuation
of inflammation. Moreover, T. cruzi requires β1 integrins
to gain access to the cell(71). An increased expression of
ICAM-1, VCAM-1, and ECM components has been detected
in heart and endothelial cells of patients with CCC(72), which
can be secondary to increased inflammation. Cytokines and
chemokines produced in response to the parasite may also
modulate VCAM-1 and ICAM-1 adhesion molecules on
endothelial cells of target tissues, which recruit VLA-4+LFA1+CD8+ T lymphocytes, the predominant subset present
in inflamed heart(73).
CCC patients also have increased expression of MHC
molecules. Class I MHC molecules are up-regulated in the
sarcolemma of cardiomyocytes and there is also evidence
for an over-expression of class II MHC on endothelial
cells(74–76). This may favour the presentation of cryptic epitopes.
In addition, the number of CD4+ T cells increases parallel
to the number of CD8+ T cells in the acute phase but not
in the chronic phase suggesting an immunological imbalance.
In the chronic phase, patients with heart failure present a
predominance of CD8+ T cells with an altered CD4+/CD8+
T cell ratio. The inflammatory response, which is probably
recurrent and undergoes periods of more accentuated
exacerbation, is most likely responsible for progressive
neuronal damage, microcirculation alterations, heart matrix
deformations and consequent organ failure.
f) Polyclonal B cell activation. A generalised activation
of B cells has been shown in animal models of infection,
but the exact mechanism for it is not clear. This may disrupt
normal immune regulatory mechanisms and can lead to
autoimmunity(77).
g) Autoimmunity. It may occur either by T. cruzi-specific
mechanisms (molecular mimicry) or by non parasite–specific
effector mechanisms, eventually leading to the development
of pathology (see Table I).
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IS THERE A PATHOGENIC ROLE OF AUTOIMMUNE RESPONSES IN CHAGAS’ DISEASE?
An important issue that is often ignored in this debated
field is that none of the mechanisms listed above is mutually
exclusive. In fact, it seems unlikely that inflammation occurs
through only one of these mechanisms.
AUTOIMMUNITY/ PARASITE PERSISTENCE DILEMMA
The finding of a T-cell rich inflammatory mononuclear
cell infiltrate and the scarcity of parasites in heart lesions
questioned the direct participation of T. cruzi in CCC and
suggested the possible involvement of autoimmunity(11).
This remains, however, a hotly debated issue(11,67,78-82). Several
early studies on Chagas’ disease already emphasised the
scarcity of parasites in histological sections in the chronic
phase of the disease(83,84). Since then, much research has
focused on the possibility that autoimmune responses set
off by molecular mimicry and/or bystander activation
contributes to tissue damage. Those mechanisms were
initially reported many years ago(85-92) and are supported
by a large body of circumstantial evidence thereafter(11,7981,93,94). All those studies have contributed to the popularity
of this hypothesis among researchers in the field.
However, mounting evidence are challenging this view.
The use of more sensitive techniques has allowed detection
of parasite antigens or DNA during the chronic phase.
Therefore, all the damage could be attributed either to an
inflammatory response against the parasite or to the parasite
replication itself(67,68,78). It should be emphasized that not a
single report published to date has unequivocally demonstrated
that either autoimmunity or parasite-specific immunity are
pathogenic.
Of the mechanisms of induction of autoimmunity shown
in table I, which ones can be found in T. cruzi infection? The
detection of circulating anti-T. cruzi antibodies that crossreact with host tissue antigens is a common finding in human
and animal models of chagasic infection. There are numerous
reports of T. cruzi antigens cross-reactive with heart and
neural tissues of the host(79,81,95). However, with few exceptions,
the autoantibodies or autoreactive T cells against those
antigens do not seem to be the leading cause of autoimmune
pathogenesis. The most relevant autoantigens involved in
pathology are described below.
Autoantibodies
Cardiac myosin is one of the favourite, and most debated,
autoantigens that have been implicated in the aetiology
of CCC. A well-studied T. cruzi candidate implicated in
pathogenesis through molecular mimicry is B13 protein
that crossreacts with human cardiac myosin. Cunha-Neto
and his collaborators have described myosin heavy chain
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as a major antigen of heart-specific autoimmunity and
suggested the possible relevance of myosin recognition in
human CCC(96,97). Antibodies to myosin were found in both
asymptomatic and chronic chagasic patients: 14/23 (61%)
symptomatic and 1/14 (7%) asymptomatic patients. Affinitypurified anti-myosin antibodies specifically recognised two
bands of 140,000 and 116,000 daltons in extracts from T.
cruzi trypomastigotes. Sera from all patients with CCC
disease recognised a recombinant T. cruzi peptide, named
B13, but only 14% of the anti-myosin positve sera from
asymptomatic chagasic patients(98). At the molecular level,
crossreactivity was shown to exist between the amino acid
sequences AAALDK and AAAGDK from cardiac myosin
and B13, respectively.
Results from the Engman’s group showed that T. cruziinfected A/J mice (a strain highly susceptible to T. cruzi
infection) generated anti-myosin IgG, both in the acute and
chronic phases of the infection(99). They also found that
immunisation with purified myosin caused some heart
lesions resembling those seen in T. cruzi-infected mice.
However, not all mouse strains are equally susceptible to
myocytolysis after T. cruzi infection(94). Interestingly, in
C57BL/6 mice, a strain more resistant to development of
myocarditis, the levels of anti-myosin IgG found after T.
cruzi infection were either small or undetectable(99). This
mouse strain has been claimed not to develop cardiac
autoimmunity after immunisation with myosin(100). However,
we have detected some signs of inflammation in T. cruzi
infected C57BL/6 mice, although in a much lesser extent
than in iNOS knockout mice (Fig. 2). Since C57BL/6 develop
lower parasitaemias, this may indicate that the presence of
myocarditis may depend on the initial level of control of
parasite replication in the acute phase.
Other reports suggested that anti-myosin antibodies
are not involved in the pathogenesis. For example
immunisation with myosin in immunosuppressed mice did
not induce the production of antibodies but still caused
myocarditis. However, how myocarditis can be triggered
by myosin in those immunosuppressed mice is difficult
to envisage. Moreover, passive transfer of a high-titre antimyosin antibody preparation failed to induce myocarditis(101).
Since the fine specificity of the different anti-myosin IgGs
has not been addressed in most of those studies, it is difficult
to compare them. It is possible that the myosin determinant(s)
recognised by the different sera are not identical.
C57BL/6 mice infected with a highly virulent T. cruzi
strain developed severe cardiomyopathy and produced
autoantibodies that recognised antigens from a mouse heart
extract(102). However, unlike Cunha-Neto et al., the authors
of this study observed no cross-reactivity between mouse
INMUNOLOGÍA
Figure 2. Induction of high parasitaemia and myocarditis in iNOS knockout
mice with a resistant genetic background. C57BL/6 and inducible nitric oxide
synthase (iNOS)-deficient mice were infected with 5x103 T. cruzi blood
trypomastigotes of the Tulahuen strain, obtained from previously infected
mice. At 14 days post infection (d.p.i.) iNOS-deficient mice showed a 200fold increase in parasitaemia respect to C57BL/6 mice (data not shown).
Haematoxylin and eosin staining of heart tissue sections at 28 d.p.i. revealed
the presence of amastigote nests only in infected iNOS-deficient mice (black
arrowheads in c and d). Moreover, an increase in mononuclear cell infiltrates
in iNOS-deficient mice respect to wild type C57BL/6 mice was observed
(white arrowheads in c and d versus a). C57BL/6 mice chronically infected
with T. cruzi (120 d.p.i.) also developed mononuclear cell infiltration (white
arrowhead in b). Magnification is indicated in each section.
anti-heart antibodies and T. cruzi epimastigote antigens.
Furthermore, sera from mice hyperimmunised with myosin
failed to recognise any antigen on T. cruzi epimastigote
extracts, and sera from mice hyperimmunized with T. cruzi
did not detect any muscle tissue antigen (102). Since these
experiments were not carried out with circulating forms in
the host (trypomastigotes or amastigotes), the results are
inconclusive, and molecular mimicry between heart and T.
cruzi antigens can not be completely ruled out. In contrast
with the above, purified anti cruzipain antibodies made
in mice cross-react with myosin heavy chain(103).
Of note, anti-myosin antibodies are also significantly
induced in patients having heart disease not related to T.
cruzi infection such as viral myocarditis, myocardial infarction,
coronary artery bypass and heart valve surgery, among
others(104-106). Thus, rather than cross-reactive T. cruzi antigens,
myocyte damage caused either by parasite replication in
N. GIRONÈS, H. CUERVO, M. FRESNO
the heart or by inflammation may release self-antigens
leading to the induction of anti-heart antibodies. It would
not seem unreasonable, therefore, to infer that the initial
insult resulting from T. cruzi infection could cause a rise
in the level of anti-myosin immunity in Chagas’ heart disease.
A criticism often raised against cross-reactive T. cruzi
proteins is that, despite evidence of immune responses
against both the parasite and the putative self-antigens,
there is no direct evidence demonstrating that they can
induce autoimmunity. However, there are increasing numbers
of reports addressing this criticism. Thus, autoimmune
response in mice immunised with cruzipain was associated
to heart conduction disturbances. In addition, ultrastructural
findings revealed severe alterations of cardiomyocytes and
IgG deposition on heart tissue of immunised mice. We
investigated whether antibodies induced by cruzipain
transferred from immunised mothers to their offsprings
could alter the heart function in the pups. All IgG isotypes
against cruzipain derived from transplacental crossing were
detected in pups' sera. Electrocardiographic studies performed
in the offsprings born to immunised mothers revealed
conduction abnormalities. These results provide strong
evidence for a pathogenic role of autoimmune response
induced by a purified T. cruzi antigen in the development
of experimental Chagas’ disease(103). Moreover, antibodies
to other cross-reactive antigens such as anti β-adrenergic(107)
and anti muscarinic receptor(108,109) that cross-react with T.
cruzi ribosomal proteins may also cause cardiac pathology.
Recently, it has been described that cruzipain also induces
autoantibodies against muscarinic acetylcholine receptors
which can be implicated in pathology(110). All those purified
autoantibodies may alter cardiac beating in the mouse heart
and explain some of the pathological findings of CCC. This
not necessarily proves molecular mimicry, since the bystander
activation mechanism for the generation of anti-self responses
cannot be ruled out.
Along those lines, some authors believe that other
mechanisms different from molecular mimicry can explain
autoreactivity(9,79). They think that mimicry is less likely to
be true than antigen release due to myocardial damage
leading to expansion of normally tolerant myosin-reactive
T cells, particularly since myosin autoimmunity is seen in
myocarditis associated with other insults. Besides, B cell
anti myosin response seems to be the main cause of pathology
in other heart infections, induced by Coxsackie B3(111) or by
bacteria(112). In this regard, it is worth mentioning that peptides
of cardiac myosin, a cytoplasmic protein, are associated
with MHC class II molecules on APC even in normal mouse
myocardium(113) and that MHC class II molecules are increased
in the heart of T. cruzi-infected patients and animals (see
191
IS THERE A PATHOGENIC ROLE OF AUTOIMMUNE RESPONSES IN CHAGAS’ DISEASE?
above). Alternatively, it is possible that some pathogens
may share the ability to destroy the heart but they may have
different cross-reactive epitopes with heart proteins (myosin).
Thus, it is possible that the trigger is the combination of
pathogen and pathogen damage, although the fine specificity
of the autoreactive response will be different in each case.
While the presence of «anti-self» immune responses in
T. cruzi infections has been unquestionably demonstrated,
evidence for the mediation of cross-reactive antibodies or
T cells in pathology is still lacking.
Autoreactive T cells
Perhaps the most compelling evidence supporting a role
for autoantigen-specific autoimmunity in the pathogenesis
of the disease derives from T cell mediated immunity. We
have shown that reactivity against a dominant autoantigen
(named Cha) in human and mice T. cruzi infections is the
result of molecular mimicry between clearly distinct T and
B cell Cha epitopes and highly immunogenic parasite antigens,
which triggers strong T and B cell-dependent responses(114).
More interestingly, adoptive transfer of autoreactive T cells
isolated from spleen of chronically infected mice induced
heart infiltrates in recipient mice and triggered autoantibody
production in the absence of the parasite. In contrast, we
were not able to induce pathology by immunising with the
Cha crossreactive antigen. Although our results suggested
that Cha might be involved in pathology, this by no means
indicates that Cha would be the only autoantigen involved
in autoimmune pathology of Chagas’ disease.
In the same direction, Ribeiro-Dos-Santos et al have
described that a CD4+ T cell line obtained from a chronic
chagasic mouse induces carditis in immunised mice and
rejection of normal hearts in the absence of T. cruzi. Therefore,
in some cases the presence of the parasite is not necessary
to produce pathology if activated autoreactive T cells are
transfered. The requirement of the parasite to cause rejection
in mice transferred with T cells from infected mice has also
been widely debated(97,115-117). Chronically infected mice cause
rejection of syngeneic transplanted hearts either in the
absence (116) or in the presence of the parasite (115). These
differences may be due to different mice and parasite strains
used, and when the presence of the parasite is required
for rejection, inflammation and not T. cruzi replication may
be necessary to provide the necessary adjuvant effect to
trigger autoreactivity and could be the rejection inducing
agent in the implanted hearts.
It has been shown that immunological tolerance to heart
antigens induced in mice by heart antigen administration
prior to their infection by T. cruzi resulted in less intense
cardiopathy than control non-tolerised animals(118). However,
192
VOL. 23 NUM. 2/ 2004
recently Leon et al. have described (although in the acute
phase) that myosin autoimmunity, while a potentially
important inflammatory mechanism in acute and chronic
infection, is not essential for cardiac inflammation(119). Thus,
it is probable that autoreactivity itself is not sufficient to
induce tissue inflammation, and that a proinflammatory
environment, induced by infection(120), is needed.
Parasite persistence
Tarleton has sequentially reviewed, ardently and somewhat
biased, all the arguments in favour of the parasite persistence
hypothesis to explain the patogenesis of chronic Chagas’
disease in general and of CCC in particular(67,68,78). Arguments
in favour that the disease is linked to parasite presence are
supported by the fact that treatments that decrease the
parasite burden in the acute phase are associated to a decrease
in the clinical symptoms(121). Effective chemotherapy could
also enhance anti-T. cruzi immunity in mice(122). In humans,
the link between persistence of T. cruzi and clinical disease
is also supported by the tissue-specific detection of parasite
DNA in the heart, but not in the oesophageal tissue, of
individuals with cardiac disease; and viceversa (123,124).
Moreover, several data support that enhancing the efficiency
of the anti-parasite response by immunotherapy, genedeletion, or vaccination, results in a decreased severity of
the chronic phase, and not exacerbation of the disease as
predicted by the autoimmune hypothesis(78). Very recent
data in humans show a very high frequency of parasitespecific IFNγ- producing CD8+ T cells among chronic patients
with mild clinical disease than in those with the most severe
form of the disease, supporting a link between the strength
and nature of the anti parasite response and the severity of
the chronic stage of the disease(125). Thus, the stronger the
immune response, the better the outcome. Conversely, it
has been observed that immunosuppressive treatments
correlate with exacerbation of the infection and disease(126).
Apparently this supports the parasite persistence hypothesis
in opposition to autoimmunity, strongly arguing in favour
of the participation of an effective antiparasite response in
preventing the disease(78). However, those reports suggest
that all the damage is parasite-mediated and could be also
taken as an argument against supporters of parasite–induced
immune response as a mediator of cardiac damage(72).
We have found that autoreactivity in the chronic phase
is also linked to the parasitaemia since the antibody titre and
reactive T cells against the Cha autoantigen are lower in
C57BL/6 (non-susceptible) than in BALB/c (susceptible)
mice (114). Moreover, potentially pathogenic anti-Cha
autoantibodies also decreased with the treatment of patients.
The titre of anti-Cha and anti-parasite antibodies decreased
INMUNOLOGÍA
N. GIRONÈS, H. CUERVO, M. FRESNO
TABLE III. Myocarditis and antibody responses in chagasic patients increase with symptomatology and decrease with treatment
Chagasic patients
Symptomatic
Asymptomatic untreated
Asymptomatic treated
Anti-Cha antibodies
Anti-T. cruzi antibodies
Myocarditis
+++
++
+
+++
++
+
+++
-
The presence or absence of myocarditis was given by clinical histories of patients.Antibody response was quantitated as reported elsewhere (127): +, OD
450 nm<0.3; ++, OD 450 nm 0.3-1.0; +++, OD 450 nm<1.0.
in parallel with treatment and increased with symptomatology(127)
(Table III).
The presence of T. cruzi in the chronic phase of the disease
was already observed in early descriptions(128) and has been
documented later by other authors(129,130). With more sensitive
techniques such as polymerase chain reaction (PCR), parasite
DNA is commonly detected in chronic patients(72). Recent
studies using modern immunohistochemistry have
demonstrated higher frequencies of T. cruzi antigens. T.
cruzi antigens were detected in 100% of hearts from chronic
chagasic patients that died from heart failure when several
samples of the myocardium were analysed(131,132). Many
previous failures to detect parasite antigens from biopsy
material of patients in the chronic phase have been attributed
to the fact that it seems necessary to examine several different
sections of the heart to detect the parasite in this phase of
the disease(72). Using a mouse strain that develops chagasic
cardiomyopathy when infected with a highly virulent T.
cruzi strain, amastigotes were detected in myocytes throughout
the chronic phase, although their numbers strongly decreased
and were scarce already in the early chronic phase(133). A
general finding not always acknowledged by the supporters
of the parasite persistence hypothesis is that there is no
direct correlation between the sites of parasite detection and
heart damage, and also no correlation between the levels
of parasites (for example as detected by PCR) and clinical
findings(134). However, a significant association between the
presence of T. cruzi antigens in the heart and severe or
moderate inflammation was observed both in humans(72)
and animal models of the disease(135). Thus, a low number
of parasites correlated with intense myocarditis and whole
myocardial fibres containing parasites did not elicit
inflammation(72). Besides, some of the work cited to support
parasite persistence could be used against it. For instance
Buckner et al said cardiac tissue had relatively dense
acute parasitism (although 100-fold less than in the acute
phase) but showed minimal inflammation in the chronic
stage. In contrast, peripheral nerve tissue had few parasites
but was heavily inflammed in the chronic stage. At the most,
inflammation in the nerve occurred in the presence of very
few T. cruzi parasites, and it possibly occurred in the presence
of none(135). This suggests two possibilities: exuberant host
reactions to the few remaining parasites, either immunemediated or not, or autoimmune-induced inflammation.
Parasite antigens probably work as a trigger response against
the myocardial fibres. It is possible that some lesions lack
parasites or parasite antigens because of the effective clearance
of parasites from this site by an effective anti-parasite immune
response, thus preventing the observation of an exact
correlation. However, as mentioned before, this is difficult
to reconcile with the fact that a strong anti-parasite immune
response results in decreased symptoms(125).
Thus, parasites are somehow present in the chronic phase
but what one ought to know is whether relevant parasite
antigens persist and are presented by APC to T cells. No
matter the antigen recognised, antigen specific T cells need
to be stimulated to become effector cells (helper, cytotoxic
or other). For this, the antigen needs to be presented. Although
some APC could be very efficient in presenting antigens it
is rather unlikely that there could be enough parasite antigens
to continuously support chronic T cell stimulation.
Demonstration of hypotheses
As mentioned above, the hypothesis of molecular mimicry
is very popular in the T. cruzi field. Several criteria(9), put
originally forth in the T. cruzi field(11) need to be met to
consider a disease as caused by molecular mimicry (Table
II). In T. cruzi infection, the first three conditions have been
clearly demonstrated, and this has allowed the identification
of several candidate autoantigens. If there were a unique
cross-reactive antigen, infection with genetically deficient
parasites lacking the inducing antigen, or infection of knockout
mice lacking the cross-reactive autoantigen, would prevent
the disease. However, as multiple autoantigens seem to
be involved in the pathology of Chagas’ disease, such
experiments are very difficult to perform, and therefore the
fourth criterion has not been demonstrated yet. The fifth
criterion is considered to be the decisive test of the concept
of autoimmunity. In this respect, the presence of autoreactive
T cells against cardiac myosin/B13 proteins in heart lesions
193
IS THERE A PATHOGENIC ROLE OF AUTOIMMUNE RESPONSES IN CHAGAS’ DISEASE?
has been described, but its contribution to the pathology
has not been demonstrated(97).
The autoimmune hypothesis for Chagas’ disease is based
on the fact that parasites are almost undetectable in the
chronic phase of the disease and because of that it postulates
that autoimmunity would be the result of an autoimmune
aggression. In most publications about autoimmunity in
Chagas’ disease the putative causes are either autoantibodies
or autoreactive T cells originated by molecular mimicry
between parasite and host antigens. Nevertheless, evidence
for the mediation of cross-reactive antibodies or T cells in
pathology is still lacking. Moreover, most of the data
come from the experimental T. cruzi infection, and an
additional problem is the extrapolation of the results to the
human model which is more difficult to study.
One way to determine the pathologic effect of autoreactive
T or B cells would be to immunise mice with cross-reactive
antigens to see if this induces pathology. However,
immunisation with an autoantigen (injected together with
adjuvants and via routes different from that of the natural
infection) may not reflect the way the autoantigen is presented
during natural infection and may elicit either hyperimmune
responses, or tolerance, or regulatory T cells that may suppress
autoimmunity. An alternative approach is the transfer of
putative autoreactive T cells from chronically infected mice
or T cells specific for a given autoantigen. Either way may
answer some of these questions and determine which of the
candidates are really relevant for pathology. Altered peptide
ligands may also be useful for treatment of the disease.
The parasite persistence hypothesis is based on the fact
that T. cruzi persists in the chronic phase of Chagas’ disease
and that treatment against the parasite results in a decrease
of the severity of the disease. A demonstration of this
hypothesis is also difficult to perform, since one ought to
separate the components of the immune response, self and
anti-self during infection. If there were a unique crossreactive antigen, gene deletion of cross-reactive antigens in
the parasite should have an impact (either positive or negative)
on the pathology. This would theoretically solve the dilemma.
However, things are not so easy, since cardiac damage and
humoral and cellular cardiac autoimmunity have been
recently reported in mice during the acute phase when
parasitaemia is high(99). Furthermore, the coexistence of self
and non-self antigens would enhance the immune response
against both, always triggered by the parasite.
There are also some questions that need to be fully
addressed: 1) why do lesions develop primarily in the heart
and not at other sites of parasite persistence?, and 2) why
does the parasite burden not always correlate with disease
severity?.
194
VOL. 23 NUM. 2/ 2004
T. cruzi
T and B cell
antigen-specific
signals
Molecular
mimicry
Autoimmune
disease
Inflammation
Adjuvant
effect
Release of
self-antigens and
cryptic epitopes
Bystander
activation
Autoimmune
disease
Figure 3. Diagram representing the different mechanisms of induction of
autoimmune disease by T.cruzi. T.cruzi can induce T and B cell anti-parasite
responses, which can induce autoimmune disease through molecular mimicry
with extracellular antigens or epitopes in antigens normally presented by
APC. T. cruzi can also induce cytokines that mediate some cardiac damage
and liberation of autoantigens recognised by autoreactive T cells and
autoantibodies that further damage the cardiac tissue via bystander activation.
Simultaneously, T. cruzi can induce release of self-antigens, usually intracellular,
which contain cryptic epitopes that can be presented by APC. Overexpression
of intracellular antigens induced by T. cruzi can also end up with presentation
of cryptic epitopes by APC. If cryptic epitopes are cross-reactive with T. cruzi
epitopes, then autoimmune disease can arise. T. cruzi contains several molecules
capable of stimulating the immune system in a non-antigen specific manner,
known as adjuvant effect, which together with the release of self-antigens
and exposure of cryptic epitopes can contribute to sustain a local immune
activation known as bystander activation.
Coexistence of parasite persistence and autoimmunity
We think that since cardiac myosin autoimmunity
develops in the acute phase, where there is lysis of cardiac
myocytes and easily detectable parasites, it is very likely
that both processes, bystander damage and molecular
mimicry coexist till the chronic phase. Then, damage goes
via effector cells which recognise crossreactive T.
cruzi/autoantigen through molecular mimicry.
Thus, we propose that parasite is the trigger that activates
some T cells (autoantigen/crossreactive parasite antigen).
Once they are activated, they secrete inflammatory cytokines
that mediate some cardiac damage. This liberates autoantigen
that is also recognised by some other autoreactive T cells
and autoantibodies that further damage the cardiac tissue
via bystander activation. This is like a vicious circle triggered
by parasite antigens but fuelled by crossreactive autoantigens
and implies that purely parasite specific T cells may cause
very little cardiac damage. This also involves two of the
proposed pathogenic mechanisms: bystander damage
and molecular mimicry. T. cruzi might also function as an
adjuvant for an immunological cross-reaction between
common parasitic and myocardial fibre antigens, resulting
in severe lymphocytic myocarditis (Fig. 3).
INMUNOLOGÍA
Thus, parasites are necessary to trigger autoantibodies
and autoreactive T cells and may be even necessary to
maintain them in the chronic phase. Nevertheless, this does
not prove that crossreactivity of those autoantibodies and
autoreactive T cells with self-components, which either are
expressed at the cell surface or presented by APC upon
infection, have no effect at all in the pathology. Altogether,
we believe that active T. cruzi infection is necessary to trigger
the autoimmune process, most likely through autoreactive
T cells, that once induced can transfer the cardiac pathology.
In summary, we believe that the debate on T. cruzi myocarditis
closely resembles the one generated by coxackie virus(1,8).
After all, the right answer may lie between both hypotheses,
pathogens (parasites, virus) are the trigger but autoreactive
T and B cells are the actual effector cells.
ACKNOWLEDGEMENTS
The experimental work of the authors mentioned in this
manuscript has been supported by grants from the following
Spanish organizations: Ministerio de Ciencia y Tecnología,
Red RICET (red de investigación de centros de enfermedades
tropicales), Fondo de Investigaciones Sanitarias, Comunidad
Autonoma de Madrid and Fundacion Ramon Areces. We
thank Gloria Escribano for her technical assistance in the
preparation of this manuscript.
CORRESPONDENCE TO:
Manuel Fresno
Centro de Biología Molecular, CSIC-UAM
Universidad Autónoma de Madrid,
Cantoblanco 28049 Madrid, Spain.
Phone: 34-91-4978413. Fax: 34-91-4974799
E-mail: [email protected]
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