[Advances in Parasitology] Chagas Disease, Part B Volume 76 || Autoimmunity

CHAPTER 6

Advances in Parasitology, VISSN 0065-308X, DOI: 10.1

* Laboratorio de ImunologUniversidade de Sao Pau

{ Disciplina de ImunologiaSP, Brazil

{ Instituto de Investigacao

Autoimmunity

Edecio Cunha-Neto,*,†,‡ Priscila Camillo Teixeira,*,‡

Luciana Gabriel Nogueira,*,‡ and Jorge Kalil*,†,‡

Contents 6.1. Introduction 130

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tituto do Coracao, Hospital das Clınicas, Faculdade de MedicinPaulo, SP, Brazil

a e Alergia, Faculdade de Medicina, Universidade de Sao Paulo

unologia—INCT, Sao Paulo, SP, Brazil

Elsets

a,

, Sa

6.2. N
atural History of Chagas Disease 134 6.3. H eart-Specific Inflammatory Lesions in CCC: Parasite
Antigen-Driven Immunopathology?
134 6.4. Im munopathogenesis of CCC 135 6.5. A utoimmunity in Chagas Disease 137
6
.5.1. A utoantibodies 138 6 .5.2. A utoreactive T cells 139
6.6. M
olecular Mimicry 140 6.7. C onclusion 142 Referen ces 144
Abstract The scarcity of Trypanosoma cruzi in inflammatory lesions of chronic

Chagas disease led early investigators to suggest that tissue damage

had an autoimmune nature. In spite of parasite persistence in chronic

Chagas disease, several reports indicate that inflammatory tissue

damage may not be correlated to the local presence of T. cruzi. A

significant number of reports have described autoantibodies and self-

reactive T cells, often cross-reactive with T. cruzi antigens, both in

patients and in animalmodels. Evidence for a direct pathogenetic role

of autoimmunity was suggested by the development of lesions after

vier Ltd.reserved.

˜o Paulo,

129

130 Edecio Cunha-Neto et al.

immunizationwithT. cruzi antigensorpassive transferof lymphocytes

from infected animals, and the amelioration of chronicmyocarditis in

animals made tolerant to myocardial antigens. Autoimmune and

T. cruzi-specific innate or adaptative responses are not incompatible

or mutually exclusive, and it is likely that a combination of both is

involved in the pathogenesis of chronic Chagas disease cardiomyop-

athy. The association between persistent infection and autoimmune

diseases—such as multiple sclerosis or diabetes mellitus—suggests

that post-infectious autoimmunity may be a frequent finding. Here,

we critically review evidence for autoimmune phenomena and their

possible pathogenetic role in human Chagas disease and animal

models, with a focus on chronic Chagas disease cardiomyopathy.

6.1. INTRODUCTION

Chronic Chagas disease Cardiomyopathy (CCC) is one of the few well-defined examples of human post-infectious autoimmunity, where aninfectious episode with an established pathogen—the protozoan parasiteTrypanosoma cruzi—triggers multiple autoimmune phenomena, mostrelated to documented molecular mimicry, and organ-specific damage.Indeed, several bona fide autoimmune diseases, like multiple sclerosis(Ablashi et al., 1998) and insulin-dependent diabetes mellitus (el-Zayadiet al., 1998), have been associated with persistent infections, which sug-gest that post-infectious autoimmune diseases are more frequent thanpreviously thought.

The timescale dissociation between primary infection with high tissueand blood parasitism and tissue pathology, allied to the scarcity of T. cruziin CCC heart lesions, prompted investigators as early as 80 years ago tosuggest that the mononuclear cell infiltrate should directly damage theheart, perhaps in an autoimmune fashion (Torres, 1929). A significantnumber of reports have described autoantibodies and self-reactive Tcells, many times cross-reactive with T. cruzi antigens, both in patientsand in animal models (summarized in Table 6.1). Evidence for a directpathogenetic role of autoimmunity was suggested by the development oflesions or functional damage after immunization with T. cruzi antigens orpassive transfer of lymphocytes and autoantibodies from infected ani-mals (summarized in Table 6.2). The amelioration of chronic myocarditisin animals made tolerant to myocardial antigens also suggested apathogenic role for autoimmunity in Chagas disease. In this chapter, wereview the evidence for the role of pathological autoimmunity in thepathogenesis of Chagas disease.

TABLE 6.1 Molecular mimicry after T. cruzi infection

Host component T. cruzi antigen Host

Molecular

definition Reference

Neurons, liver, kidney,

testis

unknown M, R Mab Snary et al. (1983)

Neurons unknown R Mab Wood et al. (1982)Neurons Sulphated glycolipids H Mab Petry et al. (1987a,b) and Petry and Eisen

(1988, 1989)

Heart tissue unknown M Serum IgG McCormick and Rowland (1989)

Heart and skeletal muscle Microsomal fraction H Mab Laucella et al. (1996a,b)

Human cardiac myosin

heavy chain

B13 protein H rDNA, Ab,

T cell clones

Cunha-Neto et al. (1995, 1996) and

Abel et al. (1997)

Human cardiac myosin

heavy chain

Cruzipain M Ab Giordanengo et al. (2002)

95 kDa myosin tail T. cruzi cytoskeleton M Mab Oliveira et al. (2001)

Skeletal muscle calcium-

dependent SRA

SRA Rb, H AS Acosta et al. (1983) and Santos-Buch et al.

(1985)

Smooth and striated

muscle

150 kDa protein H, M Serum IgG Zwirner et al. (1994)

Glycosphingolipids glycosphingolipids H, M Serum IgG Vermelho et al. (1997)

MAP (brain) MAP H, M rDNA, AS Kerner et al. (1991)

Myelin basic protein T. cruzi soluble extract M Serum IgG, Tcells

Al-Sabbagh et al. (1998)

28 kDa lymphocyte

membrane protein

55 kDa membrane

protein

H, M Mab Hernandez-Munain et al. (1992)

(continued)

TABLE 6.1 (continued )

Host component T. cruzi antigen Host

Molecular

definition Reference

47 kDa neuron protein FL 160 H rDNA, AS Van Voorhis and Eisen (1989) and

Van Voorhis et al. (1991, 1993)23 kDa ribosomal protein 23 kDa ribosomal

protein

H Ab Bonfa et al. (1993)

Ribosomal P protein Ribosomal P protein H rDNA, Ab, SP Levitus et al. (1991)

b1 adrenoreceptor M2

cholinergic receptor

Ribosomal P0 and P2bproteins

H rDNA, Ab, SP Ferrari et al. (1995), Kaplan et al. (1997),

Lopez Bergami et al. (1997, 2001),

Masuda et al. (1998) and Mahler et al.

(2001)

b1-adrenoreceptor M2cholinergic receptor

150 kDa protein H, M Mab Cremaschi et al. (1995)

M2 cholinergic receptor unknown H Ab Motran et al. (1998)

38-kDa heart antigen R13 peptide from

ribosomal protein

P1, P2

M IgG1, IgG2 Hernandez et al. (2003)

Cha antigen SAPA, 36 kDa

TENU2845

M Ab, T cell Girones et al. (2001b)

Calreticulin Calreticulin H,M Ab Ribeiro et al. (2009)

M, mouse; H, human; Rb, rabbit; R, rat; AS, antiserum; Ab, patient antibody; Mab, monoclonal antibody; rDNA, recombinant DNA; SP, synthetic peptides.

TABLE 6.2 Evidence for pathological autoimmunity in Chagas disease

T. cruzi antigen Host Effect References

Effects of immunization with T. cruzi antigens

T. cruzi microsomal fraction Rb Myocarditis Teixeira and

Santos-Buch

(1975)

T. cruzi SRA M Myocarditis Acosta and Santos-

Buch (1985)

T. cruzi microsomal andcytoplasmic fractions

M Myocarditis Ruiz et al. (1985)

Recombinant ribosomal

protein P2bM ECG

alteration

Lopez Bergami et al.

(1997)

R13 peptide from ribosomal

protein P0

M ECG

alteration

Motran et al. (1998)

Immunological effectors Host Effect References

Effects of passive transfer of antibodies or T cells from chronically

T. cruzi-infected hosts

Splenocytes M Focal myocarditis Laguens et al. (1981)

CD4þ T-cell lines M Demyelination Hontebeyrie-Joskowicz

et al. (1987)

CD4þ T splenocytes M Focal myocarditis dos Santos et al. (1992)

and Silva-Barbosa

et al. (1997)Anti-T. cruzi MAb M cAMP synthesis,

Increased cardiac

contractility

Zwirner et al. (1994)

and Cremaschi et al.

(1995)

Mouse anti-receptor

Ab

M Modulation of

calcium channels

Mijares et al. (1996)

Anti-M2 muscarinic

Ab from arrythmic

patients

H Conduction defect

in rabbit hearts

de Oliveira et al. (1997)

and Masuda et al.

(1998)Anti-M2 receptor O2

loop Ab from

Chagasic patients

H Decreased

contractility of rat

atria

Goin et al. (1991, 1994)

and Leiros et al.

(1997)

Ab against T. cruzi P

protein/b1adrenoreceptor

H Accelerate beating

on rat

cardiomyocytes

Ferrari et al. (1995) and

Kaplan et al. (1997)

Antigen Host Effect References

Effects of immunological tolerance induction with heart antigens

Myosin-enriched heart

homogenate

M Modulation of chronic

myocarditis and

fibrosis

Pontes-de-Carvalho

et al. (2002)

Myosin M Acute myocarditis was

not modulated

Leon et al. (2001,

2003)

M, mouse; Rb, rabbit; H, human; Ab, antibody; Mab, monoclonal antibody.


6.2. NATURAL HISTORY OF CHAGAS DISEASE

The high parasite load typical of the acute infection ensues a stronginnate and adaptative immune response against T. cruzi, leading to thecontrol—but not the complete elimination—of tissue and blood parasit-ism, establishing a low-grade persistent infection regardless of the clinicalprogression of the disease (Martin et al., 1987).

Chagas disease cardiomyopathy, the most clinically significant conse-quence of T. cruzi infection, is an inflammatory cardiomyopathy thatoccurs in 25–30% of patients, 5–30 years after infection. About a third ofpatients developing CCC present a particularly lethal form of dilatedcardiomyopathy, with shorter survival than idiopathic dilated cardiomy-opathy, often presenting with severe arrhythmia and heart block (Bocchiand Fiorelli, 2001; Mady et al., 1994). Five to 10 percentage of infectedpatients develop denervation of parietal smooth muscle in the oesopha-gus and colon, with clinical obstructive disease (Koberle, 1968). Cardiac ordigestive ‘syndromes’ of chronic Chagas disease may also present inisolated or overlapping forms. Sixty to 70 percentage chronicallyT. cruzi-infected individuals remain devoid of both cardiac and digestivemanifestations and are otherwise asymptomatic (also called ‘indetermi-nate’ patients). Functional damage of the autonomic nervous system isalso observed, affecting a subgroup of patients presenting the cardiac,digestive or asymptomatic forms of chronic Chagas disease (Amorim andMarin Neto, 1995).

6.3. HEART-SPECIFIC INFLAMMATORY LESIONS IN CCC:PARASITE ANTIGEN-DRIVEN IMMUNOPATHOLOGY?

The major histopathological feature attending dilated cardiomyopathy inCCC is the presence of a diffuse myocarditis, with intense heart fibredamage and significant fibrosis, in the presence of very scarce T. cruziforms (Higuchi et al., 1987; Higuchi Mde et al., 1993). Our group demon-strated a significant correlation between myocarditis and fibrosis andventricular dilation in the Syrian hamster model of CCC (Bilate et al.,2003). Since it is known that T. cruzi establishes a lifelong, low-gradeinfection, the possibility that chronic myocardial inflammation and tissuedamage in CCC are a consequence of recognition of parasite antigen ontarget tissue must be entertained (Higuchi et al., 1997; Kalil and Cunha-Neto, 1996). A direct role for heart parasitism has been proposed after theidentification of T. cruzi antigen and DNA in CCC hearts by immunohis-tochemical and PCR techniques (Higuchi Mde et al., 1993; Jones et al.,1993). In addition, T. cruzi-specific CD8þ T cells have been isolated from

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endomyocardial biopsies of a CCC patient (Fonseca et al., 2005),providing evidence for the recruitment and expansion of T. cruzi-specificT cells in the myocardium. In experimental T. cruzi infection, a higherinoculum or parasite load has been associated to more aggressive chronicheart inflammation or disease (Bilate et al., 2003; Marinho et al., 1999).

Several findings, however, fail to lend support to local recognition ofT. cruzi as the major trigger of heart tissue damage at the chronic phase ofChagas disease. Low-grade parasite persistence is universal in CCC andasymptomatic patients (Riarte et al., 1999; Sartori et al., 1998) and notlinked to the development of CCC (Britto et al., 1995; Pereira et al., 1992).Other evidence against it include: (i) T. cruzi DNA has been detected inhearts of both asymptomatic patients, just as frequently as among CCCpatients (Anez et al., 1999; Olivares-Villagomez et al., 1998); (ii) CD4þ T-cell clones obtained from the heart tissue of a CCC patient failed torecognize several recombinant and crude T. cruzi antigens (Cunha-Netoet al., 1996); (iii) low-grade parasitism associated to focal inflammatoryfoci in the absence of any organ functional damage is widespread inseveral organs apart from the heart (Barbosa and Andrade, 1984;Vazquez et al., 1993, 1996); (iv) immunohistochemistry and in situ hybri-dization failed to disclose an association between inflammatory lesionsand the presence of T. cruzi antigen or DNA in hearts from Chagas diseasepatients (Elias et al., 2003; Palomino et al., 2000). Taken together, evidencesuggests that the local presence of parasites may not be sufficient—oreven necessary—for inducing inflammatory tissue damage.

6.4. IMMUNOPATHOGENESIS OF CCC

Susceptibility factors leading 30% of T. cruzi-infected patients to developCCC are largely unknown. Since the bulk of evidence indicates that theinflammatory infiltrate is a significant effector of heart tissue damage, wewill review the effect of cytokines and chemokines in the pathogenesis ofCCC.

Chronic infection with T. cruzi induces a systemic shift in the periph-eral blood mononuclear cell (PBMC) cytokine profile towards Th1 cyto-kines, with suppression of Th2 cytokines (Abel et al., 2001; Cunha-Netoet al., 1998b; Gomes et al., 2003; Ribeirao et al., 2000). PBMC from CCCpatients displays an increased production of IFN-g by T cells (Abel et al.,2001; Gomes et al., 2003) or CCR5þ CXCR3þ CD4þ and CD8þ T cells, ascompared to asymptomatic patients (Gomes et al., 2005). This has beenlinked to decreased production of IL-10 (Gomes et al., 2003). In addition,PBMC from CCC patients displays a reduced number of IL-10-producingCD4þCD25high regulatory T cells and CD4þCD25highFoxP3þ regu-latory T cells, as well as increased numbers of CD4þCD25þCTLA4þ


regulatory T cells (Araujo et al., 2007) as compared to PBMC from patientsin the asymptomatic form of Chagas disease. This is in line with thefinding of increased numbers of FoxP3þmononuclear cells in myocardialsections from asymptomatic as compared to CCC patients (de Araujoet al., 2011). All chronically T. cruzi-infected patients, even from theasymptomatic form, display increased plasma levels of TNF-a as com-pared to seronegative individuals. Further, patients displaying severeCCC present significantly higher plasma levels of TNF-a and CCL2(Ferreira et al., 2003; Talvani et al., 2004). The proinflammatory and Th1-type cytokine profile described above among chronically T. cruzi-infectedpatients may be related to the ability of molecules from persisting T. cruziparasites to stimulate strong innate immunity and continuously inducethe production of IL-12 and other proinflammatory cytokines (Camargoet al., 1997). It has recently been reported that later stages of CD4þ T-celldifferentiation are associated with more severe stages of Chagas disease,suggesting that chronic T. cruzi infection might exhaust long-lived mem-ory T cells (Albareda et al., 2009).

The inflammatory infiltrate of CCC heart lesions is composed bymacrophages (50%), T cells (40%; 2:1 predominance of CD8þ overCD4þ T cells) and B cells (10%) (Higuchi Mde et al., 1993; Milei et al.,1992). CD8þT cells inCCCheart tissuewere found to expressGranzymeA(Reis et al., 1993a). The demonstration of restricted heterogeneity of T-cellreceptor Va transcripts in heart biopsies from CCC patients (Cunha-Netoet al., 1994) is in line with similar findings in established auto-immune diseases (Heber-Katz and Acha-Orbea, 1989).

Heart-infiltrating mononuclear cells predominantly produce IFN-gand TNF-a, consistent with the peripheral cytokine profile (Abel et al.,2001; Reis et al., 1993a, 1997); expression of the cytokines IL-4, IL-6, IL-7and IL-15 has also been described (Fonseca et al., 2007; Higuchi Mde et al.,1993; Reis et al., 1993a, 1997). Accordingly, CCC heart tissue also displaysincreased expression of adhesion molecules, HLA class I and class IImolecules (Reis et al., 1993b). Recent studies have shown that FoxP3þcells are significantly more abundant in myocardial sections from asymp-tomatic than in CCC patients or in infected individuals, suggesting thatregulatory T cells are less abundant in CCC than in asymptomatic hearts(de Araujo et al., 2011). In addition, increased expression of mRNA forchemokines CCL2/MCP-1, CXCL10/IP-10 and CXCL9/MIG as well astheir receptors CCR2 and CXCR3 was observed in CCC heart tissue,(Cunha-Neto et al., 2005), consistent with chemokine-driven migrationof monocytic and Th1 T cells to the CCC heart. Gene expression profilingof CCC myocardial tissue showed that 15% of genes known to be selec-tively upregulated in CCC are IFN-g inducible (Cunha-Neto et al., 2005).Moreover, exposure of neonatal murine cardiomyocytes to IFN-g upre-gulates expression of atrial natriuretic factor (Cunha-Neto et al., 2005), a

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marker of cardiomyocyte hypertrophy and heart failure. Together, theseobservations suggest that IFN-g-mediated chronic myocardial inflamma-tion could contribute to the pathogenesis of CCC, both by eliciting directinflammatory damage and by modulation of cardiac cell gene expression.

Mechanisms underlying differential progression to CCC by only 30%of chronically T. cruzi-infected patients are still incompletely understood.Familial aggregation of CCC cases has been described (Zicker et al., 1990),suggesting the existence of a genetic component in susceptibility. Associ-ation of polymorphic markers of innate immunity genes such as TNF-a,lymphotoxin-a, MAL/TIRAP (an adaptor protein involved in the TLR2-and TLR4-signalling pathway), BAT1 (an inhibitor of inflammatory cyto-kines), NFKBIL1 (potential inhibitor of NFKB) and CCL2 with CCC hasbeen reported (Ramasawmy et al., 2006a,b, 2008, 2009, reviewed inCunha-Neto et al., 2009). Further, we have shown that severe CCCpatients carrying the high TNF-a expresser genotype have shortersurvival (Drigo et al., 2006). Identification of key genes and potentgenetic combinations coupled with environmental factors may lead tothe identification of T. cruzi-infected individuals that will progressto CCC.

6.5. AUTOIMMUNITY IN CHAGAS DISEASE

The observation that most tissue pathology occurs many years after acuteinfection, when parasites were very scarce in tissue, led investigators asearly as 80 years ago (Torres, 1929) to suggest that the mononuclear cellinfiltrate should directly damage the heart, perhaps in an autoimmunefashion. Early studies were characterized by the lack of molecular defini-tion of the antigen systems employed; most used tissue or T. cruzi homo-genates. Peripheral T cells from experimentally infected mice and CCCpatients displayed responses against cardiac tissue homogenate (de laVega et al., 1976; Gattass et al., 1988). Non-infected cardiomyocyteswere targets of cytotoxicity by PBMC from chronically infected rabbits(Santos-Buch and Teixeira, 1974) and CCC patients (Teixeira et al., 1978).Repeated injection of T. cruzi subcellular fractions induced myocardialinflammatory lesions in mice and rabbits (Acosta and Santos-Buch, 1985;Teixeira and Santos-Buch, 1975).

Cossio et al. (1974) described antibodies binding to vascular endothe-lium and interstitium in mice in the serum of CCC patients, that could beabsorbed with T. cruzi epimastigotes (Table 6.1), but these were found tobe antibodies against a-galactosyl moieties, structures present in rodent,but not in human tissue (Khoury et al., 1983). Experimentally infectedmice frequently developed T. cruzi-heart muscle cross-reactive antibodies(Laucella et al., 1996b; McCormick and Rowland, 1989). Conversely,


mice with experimental autoimmune myocarditis induced by immuniza-tion with heart homogenate developed anti-T. cruzi antibodies (Chamboet al., 1990).

Several mechanisms have been suggested to play a role in the trigger-ing of autoimmunity after infection. The three mechanisms describedbelow have been demonstrated in Chagas disease patients or murinemodels and could generate experienced, effector autoreative T or B cellscapable of inducing tissue damage.

(i) Antigen exposure. T. cruzi infection promotes tissue damage and aconsequent exposure of intracellular proteins, along with activation ofinnate immunity andmyocardial inflammatory response during the acuteand chronic phases of infection, with upregulation of MHC class I andclass II proteins (Reis et al., 1993b). Self-epitopes may be presented bytissue dendritic cells in the context of MHC and upregulated costimula-tory molecules (Smith and Allen, 1992). T-cell sensitization to cardiacmyosin has been shown to occur during acute T. cruzi infection (Leonet al., 2001).

(ii) Molecular mimicry. T and B cells recognize parasite antigens thatpresent molecular mimicry with antigenically similar epitopes in hostantigens, generating cross-reactive autoimmune responses.

(iii) Polyclonal activation. Acute murine T. cruzi infection inducesantibody production that lacks a T. cruzi specificity and includesself-antigens, suggesting polyclonal B cell activation (Minoprio et al.,1988). The T. cruzi-secreted protein TcPA45 has been described as aT cell-independent B cell mitogen in mice (Minoprio, 2001).

6.5.1. Autoantibodies

During T. cruzi infection, mice can display antibodies specific for variousautoantigens contained in target tissues. Chronically T. cruzi-infectedmice display anti-tubulin IgG antibodies (Ternynck et al., 1990). Serafrom acutely or chronically infected mice recognized cardiac myosin,desmin and actin (Leon et al., 2001; Tibbetts et al., 1994). In human Chagasdisease, there is a net loss of neurons from the autonomic system along thehollow viscerae and the heart (Koberle, 1968), and sera from over 80% ofChagas disease patients contained anti-neuron autoantibodies (Ribeirodos Santos et al., 1979). Antibodies against sciatic nerve homogenatehave been found in sera from Chagas disease patients (Gea et al., 1993).Antibodies against ribonucleoproteins (Bach-Elias et al., 1998) have beendetected during T. cruzi infection. Autoantibodies against galectin-1 arecorrelated with the severity of cardiac damage in CCC (Giordanengoet al., 2001). The Cha human autoantigen and its major B cell epitopeCha are recognized by sera from Chagas disease patients (Girones et al.,2001a,b).

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Agonistic antibodies against adrenergic G-protein-coupled receptorsand the second loop of muscarinic (M2) cholinergic receptors have beendescribed (Borda et al., 1984; Goin et al., 1991, 1994, 1997; Sterin-Bordaet al., 1991). Sera from CCC patients interfere with electric andmechanicalactivities of embryonic myocardial cells in vivo (Costa et al., 2000; Kaplanet al., 1997), and sera from CCC patients induce arrhythmia in rabbithearts (de Oliveira et al., 1997). Anti-muscarinic receptor antibodies andabnormal vagal modulation occur early in Chagas disease patients, inde-pendently of the presence of left ventricular dysfunction (Ribeiro et al.,2007). Chagas disease patients showing colonic denervation syndromedisplay agonistic anti-M2 muscarinic cholinergic receptors (Sterin-Bordaet al., 2001). Differential patterns of autoantibodies towards cardiovascu-lar receptors have been associated to CCC, asymptomatic and megacolonChagas patients (Wallukat et al., 2011). The presence of such agonisticanti-receptor antibodies does not correlate with heart symptomatologybut rather with dysfunction of the autonomic nervous system (Goin et al.,1997). However, the pathogenic potential of non-functional (i.e. non-ago-nistic) autoantibodies is still a matter of debate. Complement C5–C9membrane attack complexes were found in membranes of cardiomyocytefrom CCC heart tissue (Aiello et al., 2002), suggesting that complementactivation—perhaps induced by autoantibodies—could play a role inheart tissue damage.

6.5.2. Autoreactive T cells

The first evidence for the T-cell recognition of a defined heart-specificautoantigen was provided by Rizzo et al. (1989), who showed that CD4þT cells from chronically T. cruzi-infected mice proliferated in vitro in thepresence of syngeneic cardiac myosin. Acutely T. cruzi-infected micedeveloped delayed-type hypersensitivity response against cardiac myo-sin and displayed intense myocarditis (Leon et al., 2001); anti-myosinautoimmunity was found not to be essential for acute T. cruzimyocarditis(Leon et al., 2003). Induction of tolerance with a myosin-enriched cardiachomogenate plus anti-CD4 antibody prior to T. cruzi infection resulted inreduction of chronic myocarditis and fibrosis when compared to non-tolerized infected mice (Pontes-de-Carvalho et al., 2002). Myosin is themost abundant heart protein, making up to 50% of muscle protein byweight (Harrington and Rodgers, 1984). It is a major antigen in severalinstances of heart-specific autoimmunity (Caforio et al., 1992;Cunningham et al., 1997; Neu et al., 1987b; Vashishtha and Fischetti,1993); moreover, immunization with cardiac myosin in completeFreund’s adjuvant induces severe T-cell-dependent myocarditis in genet-ically susceptible mice (Liao et al., 1993; Neu et al., 1987a, 1990; Smith andAllen, 1991). A recent study has shown that T cells from patients with the


gastrointestinal form of Chagas disease recognize an epitope in myelinbasic protein, suggesting this could be the target of autoimmunity leadingto denervation characteristic of the megacolon/oesophagus (Oliveiraet al., 2009).

Passive transfer of lymphoid cells can validate the pathogenic role ofautoimmune T cells. The transfer of T-cell populations from chronicallyinfectedmice to nerve sheaths of naıve syngeneic recipients induced nerveinflammatory lesions (Hontebeyrie-Joskowicz et al., 1987). Moreover,injection of CD4þ T cells from BALB/c mice chronically infected with T.cruzi adjacent to newborn syngeneic hearts that had been grafted intonaıve BALB/c recipients resulted in complete rejection of the transplantedheart (dos Santos et al., 1992), in the absence of T. cruzi DNA (Mengel andRibeiro-dos-Santos, 1998). Using a similar system, but employing differentT. cruzi-mouse strain combinations, another group reported that inflam-mation could only be found in the presence of T. cruzi (Tarleton et al.,1997). A heart-specific CD4þT-cell line from a chronicallyT. cruzi-infectedmouse induced death of embryonic cardiac cells in vitro (Ribeiro-Dos-Santos et al., 2001). Transfer of this T-cell line to BALB/c nude micesimultaneously immunized with syngeneic heart homogenates resultedin intense myocarditis (Ribeiro-Dos-Santos et al., 2001). Adoptive transferof splenic T cells from chronically infected mice to naıverecipients induced myocarditis in the latter and triggered antibodyresponse against the Cha autoantigen (Girones et al., 2001b). Passivetransfer and tolerance induction experiments are summarized in Table 6.2.

6.6. MOLECULAR MIMICRY

There have been several reports of immunological cross-reactivity/anti-genic mimicry between more or less defined T. cruzi and host self-anti-gens (summarized in Table 6.1). To determine whether exposure toT. cruzi antigens alone in the absence of active infection is sufficient toinduce autoimmunity, Bonney et al. immunized mice with heat-killedT. cruzi (HKTC). This immunization was capable of inducing acute car-diac damage, associated with the generation of polyantigenic humoraland cell-mediated autoimmunity with similar antigen specificity to thatinduced by infection with T. cruzi (Bonney et al., 2011). Antibodies recog-nizing calcium-dependent ATPase from the heart muscle sarcoplasmicreticulummembranes (SRA—sarcoplasmic reticulum antigen) cross-reac-tively recognized microsomal membranes from T. cruzi (Acosta et al.,1983). Immunization with T. cruzi calreticulin induces antibodies thatrecognize human and murine heart calreticulin and induces focal inflam-matory heart infiltrates (Ribeiro et al., 2009).

Cross-reactive neuron-T. cruzi antibodies have been frequentlydescribed, as displayed in Table 6.1 (Petry et al., 1987a; Snary et al.,

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1983; Wood et al., 1982). Sulphated glycolipids and neutral glycosphingo-lipids found in T. cruzi are essentially the same as found in mammalianhosts and are cross-reactively recognized by antibodies formed alonginfection (Petry and Eisen, 1989; Vermelho et al., 1997). Administrationof monoclonal antibodies cross-reactively recognizing sulphated glycoli-pids in T. cruzi and neurons induced immediate paralysis and deathby respiratory insufficiency (Petry and Eisen, 1989; Petry et al., 1988).A cross-reactive epitope was identified between T. cruzi FL-160, and aneuronal 47 kDa protein (Van Voorhis et al., 1991, 1993), but the autoanti-body failed to correlate with any clinical form of Chagas disease (Cetronet al., 1992). Cross-reactivity between T. cruzi and myelin basic proteinwas observed at the level both of antibodies and T cells in experimentallyinfected mice (Al-Sabbagh et al., 1998) (Table 6.1). Sera from T. cruzi-infected mice and Chagas disease patients contained cross-reactive anti-bodies recognizing microtubule-associated proteins from T. cruzi andfibroblasts (Kerner et al., 1991). Sera from CCC patients possessed anti-bodies against a C-terminal epitope of T. cruzi ribosomal P2b proteinwhich is conserved in mammalian ribosomal P protein (Levin et al.,1989; Levitus et al., 1991). Agonistic anti-b1-adrenergic and M2 musca-rinic receptors cross-reactive with different T. cruzi antigens werereported (Cremaschi et al., 1995; Ferrari et al., 1995; Kaplan et al., 1997;Masuda et al., 1998). Immunization of mice with T. cruzi ribosomal pro-tein P2b (Lopez Bergami et al., 1997) and the R13 peptide from ribosomalP protein (Motran et al., 1998) induced electrocardiographic alterations, inthe absence of myocardial inflammation (summarized in Table 6.2).

Cardiac myosin is a target of T cell and antibody recognition by acuteand chronically T. cruzi-infected mice (Iwai et al., 2001; Rizzo et al., 1989;Tibbetts et al., 1994). Myosin-specific delayed-type hypersensitivityresponse could be induced in mice by immunization with protein extractof T. cruzi, in the absence of detectable cardiac damage, suggestive ofcross-reactivity between cardiac myosin and T. cruzi antigens (Leon et al.,2004). Mice immunized with cruzipain, a major cystein protease fromT. cruzi, devoid of enzymatic activity developed cross-reactive anti-car-diac myosin heavy chain autoantibodies, electrocardiographic conduc-tion disturbances and myositis (Giordanengo et al., 2000a,b).

Cunha-Neto et al. (1995) detected anti-human ventricular cardiacmyosin heavy chain IgG antibodies in similar levels among sera fromindividuals in the CCC, asymptomatic and healthy soronegative subjects(Cunha-Neto et al., 1995). Affinity-selected anti-human ventricular car-diac myosin heavy chain antibodies from Chagas disease patients seraspecifically recognized a defined T. cruzi antigen (Cunha-Neto et al.,1995), the recombinant tandemly repetitive protein B13 (Gruber andZingales, 1993) (Table 6.1). Cardiac myosin-B13 cross-reactive antibodieswere predominantly found in sera from CCC rather than asymptomaticpatients (Cunha-Neto et al., 1995). CD4þ T-cell clones expandedfrom heart tissue of a CCC patient in the absence of exogenous antigen


cross-reactively recognized cardiac (but not skeletal) myosin heavy chainand T. cruzi protein B13 (Table 6.1; Cunha-Neto et al., 1996). However,in vitro sensitization of lymphocytes from a T. cruzi seronegative individ-ual with T. cruzi B13 protein or its peptides elicited B13-cardiac myosin-cross-reactive T-cell clones (Abel et al., 1997; Cunha-Neto et al., 1998a; Iwaiet al., 2005). The T-cell response to B13 protein was restricted to HLA-DR1,HLA-DR2 andHLA-DQ7, and B13 peptideswere able to bind to theseHLAmolecules (Abel et al., 2005). A B13 peptide-specific T-cell clone was estab-lished from an HLA-DQ7 individual, that cross-reactively recognizedcardiac myosin b chain peptide (5–19). Although only 5 of 15 amino acidsresidues were homologous between two peptides, amino acid scanninganalysis and molecular modeling of HLA-DQ7:peptide complexes indi-cated that TCR-exposed side chains in the cardiac myosin and B13 peptidewere almost identical (Abel et al., 2005; Iwai et al., 2005). In addition, weidentifiedmultiple very low homology cross-reactive epitopes between B13protein and human cardiac myosin (Iwai et al., 2005). The recognition ofmultiple low-homology, cross-reactive epitopes in a single autoantigenicprotein indicates intramolecular degenerate recognition which may poten-tially increase the magnitude and frequency of occurrence of the T-cell-driven autoimmune response in CCC and other autoimmune diseases.This leads to the hypothesis that in vivo sensitization with B13 antigenalong T. cruzi infection could break immunological tolerance towardscardiac myosin and elicit cardiac myosin-responsive T cells in vivo.

6.7. CONCLUSION

Chagas disease is a conundrum of several clinical syndromes triggered byT. cruzi infection in a group of susceptible individuals. Expression ofclinical syndromes can be non-overlapping. It is therefore not surprisingthat several different systems of molecular mimicry have been identified.Inasmuch as several of the cross-reactive immune responses may besimply secondary to sequence conservation or degeneracy in immunerecognition (Mason, 1998), and thus being inconsequential to pathogene-sis, it is likely that some instances of cross-reactive recognition may playan important pathogenetic role. Several reports displayed in Table 6.2 thatmatch criteria for pathologic autoimmunity (Rose and Bona, 1993) havebeen identified as follows: (i) the identification of T-cell cross-reactiveantigens, with reproduction of pathobiological changes by passive trans-fer in murine models in the absence of T. cruzi parasites; (ii) the ameliora-tion of inflammation as a consequence of tolerance induction tomyocardial antigens; and (iii) the induction of cross-reactive autoimmu-nity and end-organ dysfunction after immunization with T. cruzi or heartantigens. The isolation of T. cruzi-heart antigen cross-reactive T cells from

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myocardial tissue of CCC patients is considered important indirect evi-dence for pathological autoimmunity. It is likely that the persistence of aparasite which induces strong innate immunity and proinflammatorycytokines may continuously boost the production of potentially patho-genic Th1 T cells cross-reactively recognizing T. cruzi and heart-specificepitopes. Such Th1 T cells may migrate to heart tissue in response tolocally expressed CXCR3 ligand ckemokines. Once they reach myocardialtissue, cross-reactive T cells could be activated by cardiac antigen even inthe absence of T. cruzi antigens. This would elicit local production of Th1cytokines. Local production of Th1 cytokines could exert their pathophys-iological role by causing direct inflammatory damage, as well as modu-lating cardiac cell gene expression. Functional agonistic autoantibodiesdirected against adrenergic or cholinergic receptors may also have animportant role on autonomic system disorders and play a role in heartconduction disorders and arrhythmias. Genetic polymorphisms ofimmune response genes may affect recognition, migration and effectorcharacteristics of autoreactive T cells and autoantibodies. Finally, it mustbe stressed that autoimmune and T. cruzi-specific innate or adaptativeresponses are not incompatible or mutually exclusive, and it is likely thata combination of both is involved in the pathogenesis of CCC (Fig. 6.1).

Acute infection

Antigen exposure Molecular mimicry Polyclonal activation

T. cruzi

T. cruzi persistence

Loss of tolerance to heart antigen

Genetic susceptibility(SNPs)

Functionalautoantibodies

Pathogenicautoreactive T cells

Innateimmunity

Chronic chagas disease cardiomyopathy

FIGURE 6.1 Potential role of autoimmunity in the pathogenesis of chronic Chagas

disease cardiomyopathy.


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