Rheumatic Fever and Rheumatic Heart Disease: Genetics and Pathogenesis

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1 REVIEW doi: /j x Rheumatic Fever and Rheumatic Heart Disease: Genetics and Pathogenesis L. Guilherme*,, R. Ramasawmy*, & J. Kalil*,,à *Heart Institute (InCor), School of Medicine, University of São Paulo; Institute for Immunology Investigation, Millenium Institute; and àclinical Immunology and Allergy, Department of Clinical Medicine, University of São Paulo, School of Medicine, São Paulo, Brazil Received 13 April 2007; Accepted in revised form 19 May 2007 Correspondence to: L. Guilherme, Laboratório de Imunologia, Instituto do Coração (InCor), HC-FMUSP. Av. Dr Eneas de Carvalho Aguiar, 44-9 andar São Paulo, SP, Brazil. Abstract Molecular mimicry between streptococcal and human proteins is considered as the triggering factor leading to autoimmunity in rheumatic fever (RF) and rheumatic heart disease (RHD). Here, we present a review of the genetic susceptibility markers involved in the development of RF RHD and the major immunopathological events underlying the pathogenesis of RF and RHD. Several human leucocyte antigen (HLA) class II alleles are associated with the disease. Among these alleles, HLA-DR7 is predominantly observed in different ethnicities and is associated with the development of valvular lesions in RHD patients. Cardiac myosin is one of the major autoantigens involved in rheumatic heart lesions and several peptides from the LMM (light meromyosin) region were recognized by peripheral and intralesional T-cell clones from RF and RHD patients. The production of TNF-a and IFN-c from heart-infiltrating mononuclear cells suggests that Th-1 type cytokines are the mediators of RHD heart lesions while the presence of few interleukin-4 producing cells in the valve tissue contributes to the maintenance and progression of the valvular lesions. Introduction Rheumatic fever (RF) is a delayed sequel to throat infection by Streptococcus pyogenes and affects susceptible untreated children. Based on the major criteria established by Jones and revised by the American Heart Association, [1] the disease manifests as polyarthritis, carditis, chorea, erythema marginatum and or subcutaneous nodules. Nearly 75% of affected children display arthritis and 30 45% develop carditis, which causes heart damage with pericardial, myocardial and endocardial involvement followed by progressive and permanent valvular lesions leading to rheumatic heart disease (RHD). Sydenham s chorea is characterized by involuntary movements, especially, of the face and limbs, muscular weakness, and disturbances of speech, gait and voluntary movements. Children usually exhibit concomitant psychological dysfunction, especially, obsessive-compulsive disorder, increased emotional lability, hyperactivity, irritability and age-regressed behaviour. It is usually a delayed manifestation, and is often the sole manifestation of acute rheumatic fever (ARF) [1]. Life-threatening complications from RHD include valvulitis. Valvular lesions and mitral and aortic regurgiation are the most common events caused by valvulitis leading to chronic RHD. RF RHD is still a major public health burden in developing countries, leading to 233,000 deaths annually [2]. The incidence of RHD in the world is at least 15.6 million cases and the highest documented prevalence of the disease among children from developing countries is 5.7 per 1000 in sub-saharan Africa [2]. In Brazil, the incidence of ARF was 20,000 new cases in 1992; however, in the last 10 years it decreased by 75% but was still high, reaching 5000 new cases in 2002 (data from the Brazilian Health Ministry). The pathogenesis of RF RHD is complex and both environmental and genetic factors contribute to its aetiology. The manifestation of the disease in only a small subset of children untreated for strep throat and also the fact that only one-third of the affected children progress to the development of RHD suggests the involvement of host genetic factors. Furthermore, familial clustering and high concordance of RF RHD among monozygous twins provide conclusive evidence for the presence of genetic determinants of susceptibility to RF RHD. Nonetheless, the presence of different clinical manifestations 199

2 200 Rheumatic Fever and Rheumatic Heart Disease L. Guilherme et al. of RF RHD may also reflect extensive genetic heterogeneity. In this review, we will highlight the genetic influences involved in the pathogenesis of RF RHD as well the immunological mechanisms involved in the development of rheumatic heart lesions. Family studies in RF RHD The observation that RF clusters in families by Cheadle in 1889 led to the search for the mode of inheritance in the early 20th century and later, for the host determinants of susceptibility to the disease. Several studies suggested a simple recessive model of distribution of cases among children in rheumatic families [3, 4] while other studies revealed that there is no clear mode of inheritance [5, 6]. Despite the controversy in the mode of inheritance, these studies provide evidence that RF RHD occurs in genetically predisposed individuals. A twin study revealed a higher concordance rate (18.7%) in monozygotic than in dizygotic (2.5%) twins, suggesting very low penetrance of RF [7]. Also, the similarity of clinical manifestations in siblings of patients with RF was observed to be higher than would be expected by chance [7]. Genetic influence of the major histocompatibilty complex In an effort to identify gene(s) responsible for the development of RF, many studies were based on the presence or absence of selected blood groups (ABO) and the secretor status (ABH) of patients with RF [8 10]. Several studies have sought an association of RF RHD with either human leucocyte antigen (HLA)-A or -B loci of the major histocompatibility complex (MHC) [11 17]. Controversial results have been obtained with some finding a borderline association. The major role of HLA class I antigens is to present intracellular antigens (self and intracellular pathogen antigens) to the T-cell receptor (TCR). RF RHD, as mentioned before, is a consequence of untreated infection caused by S. pyogenes. Extracellular antigens mainly use MHC class II antigens to activate the adaptive immune response. This may be one explanation for the absence of HLA class I association with RF RHD. It is interesting to note that in 1975, HLA class II was not known, but an association with HLA-B5 and an increased response in vitro to streptococcal antigens [18] were found. A significantly more pronounced immune response by RF RHD patients as measured by circulating immune complexes [19] was also found. In both cases, the reactivity was probably due to an association with HLA class II. Strong support for this idea came from studies by Patarroyo et al. [20]. They observed that 72% of patients with RF from Bogotá, Colombia or New York, USA presented a B-cell alloantigen designated 883 [20]. The alloantigen 883 was suggested to be related to the HLA class II molecules located in the HLA-DR region of the MHC. A monoclonal antibody was produced against B cells from patients harbouring the 883 alloantigen; however, this antibody recognizes another antigen named D8 17, which is expressed on the surface of 10 20% of B cells of RF patients. The D8 17 antigen was identified in 90 95% of RF patients from different regions [21]. From this followed an investigation of HLA class II molecules in different populations. The presence of an association between HLA class II alleles and RF RHD was first described by Ayoub et al. [22], who found an association between HLA-DR2 in African-American patients and HLA-DR4 in Caucasian-American patients [22]. The association with DR4 in Caucasian-American patients was confirmed by Anastasiou-Nana [23]. HLA-DR4 was also found in Saudi Arab patients [24] (Table 1). The most consistent HLA class II allele associated with RF RHD is HLA-DR7. In Brazilian- Mullatos patients, HLA-DR7 and HLA-DRw53 were revealed to be markers for susceptibility to RF and RHD [25, 26]. In Brazilian-Caucasians, HLA-DR7 was further confirmed [27] (Table 1). It is important to note that HLA-DRw53 is of broad specificity and is associated with HLA-DR4, -DR7, and, -DR9. HLA-DR7 was also found in other populations, such as Turkish [28, 29], Latvian (in which the association of HLA-DRB1*07 and DQB1*0302 or DQB1* was also associated with the development of valvular lesions [30]), and also in RHD patients from Northern Egypt with severe mitral valve disease [31] (Table 1). Other HLA class II alleles (HLA-DR and HLA-DQ) were also found in association with RF RHD in different populations [32 39] as shown in Table 1. The strength of correlation of HLA class II alleles ranges from 2.3 times to 13.6 (RR = ) (Table 1) indicating that these alleles play a key role in the development of the disease. The fact that different class II alleles were found associated with the disease could be due to the following aspects: (1) ethnic differences of the populations studied; (2) selection of RF patients with defined clinical manifestation of the disease; (3) methodology of the study (serological or molecular biology approach); (4) size of RF RHD patient and control samples; or (5) specific streptococcal strains in different regions. Genetic influence of non-mhc Considering that both innate and adaptive immune responses are involved with the development of RF RHD, non-mhc genes probably increase the susceptibility to RF in combination with the HLA molecules. Identifying these genes will be informative in understanding the pathogenesis of the disease.

3 L. Guilherme et al. Rheumatic Fever and Rheumatic Heart Disease 201 Table 1 HLA class II and RF RHD. Country Population HLA risk Clinical picture of disease RR Ref. South África African DR1, DR6 RF RHD Martinique Admixed DR1 RHD 32 Brazil Mulatto DR1 Sydenham s chorea 34 USA African-American DR2 RF RHD Índia Indian DR3, DQW2 a RF RHD 3.76 a 35, 36 USA Caucasian-American DR4 RF RHD USA Caucasian-American DR4 RF RHD Saudi Arábia Arabians DR4 RF RHD Turkey Turkish DR11 RF RHD 37 Turkey Turkish DR3, DR7 RHD 28 Brazil Mulatto DR7, DR53 RF RHD Brazil Mulatto Allogenotope TaqI DRb kb b RF RHD 26 Brazil Caucasian DR7 RF RHD Egypt Egyptian DRB1*0701 DQ A1*0201 RHD MVR DRB1*13 DQA1*0501, DQ A1*0301 RHD Latvia Latvian DRB1*0701; DQB1*0302 RF RHD MVL DRB1*0701; DQB1*0401 RF MVR, Syndeham s chorea Turkey Turkish DRB1*07 RHD Japan Japanese DQA1*0104, DQB1*05031 RHD Mitral stenosis Mexico Mestizo DRB1*1602, DQA1*0501,DQB1*0301 RHD HLA, human leucocyte antigen; RF, rheumatic fever; RHD, rheumatic heart disease; MVL, multivalvular lesions; MVR, mitral valve regurgitation; RR, relative risk. Studies from Egypt, Japan, Latvia and Mexico employed molecular methods (PCR). a DQw2 RR value. b Defined by RFLP study, kb fragment corresponds to the HLA-DR53 antigen. ( ), not done. Mannose binding lectin (MBL) is an acute phase inflammatory protein and functions as a soluble pathogen recognition receptor. MBL binds to a wide variety of sugars on the surface of pathogens and plays a major role in innate immunity due to its ability to opsonize pathogens, enhancing their phagocytosis and activating the complement cascade via the lectin pathway [40]. MBL is encoded by the gene MBL2 located on the chromosome 10q11.1-q21 region. Mutations in exon 1 of the MBL2 gene correlate with deficient MBL in the plasma and have been shown to be associated with recurrent infections in children and several infectious diseases [40]. N-acetylglucosamine, present in the cell wall of streptococcus, is a strong ligand for MBL. Recently, one study reported that genotypes correlated with high MBL were associated with RHD [41]. Toll-like receptors are also pathogen recognition receptors that sense invading pathogens to initiate innate immune responses [42]. The search for polymorphisms in TLR related to bacterial infections is of interest in RF RHD. Berdeli et al. [43] reported a very strong association of a non-synonymous polymorphism Arg753Gln in the gene encoding TLR2 with RF among Turkish children. However, a second study in this population could not confirm this association [44]. The discrepancy between the two studies may be due to an error in genotyping or it is a reflection of distinct populations. Among Brazilian patients with RF RHD, we did not observe any association and the frequency of the mutated allele is very low in the population (R. Ramasawmy, K. C. Faé, A. C. Tanaka, G. Spina, A. C. Goldberg, J. Kalil, L. Guilherme, unpublished observations). There are many genes with inflammatory function and one of these is the interleukin (IL)-1 gene cluster that is located on chromosome 2 and includes the genes expressing the proinflammatory cytokines IL-1a and IL-1b and their inhibitor IL-1 receptor antagonist (IL-1RA). One study from Taiwan reported that variations in IL-1b and IL-1RA are not associated with RHD [45]. Further studies are warranted in other populations with larger sample size before excluding IL-1 as a susceptible or protective factor. Tumour necrosis factor A(TNFA) is another gene with inflammatory function which is situated in the MHC class III regions. Interestingly, three independent studies have shown an association of TNFA polymorphism with RF RHD. In the Brazilian population, the presence of either one of the TNFA alleles ()308A and )238A) was associated with the development of RF and RHD and this association was stronger in patients with aortic valve lesions [46]. In Turkish and Mexican populations, the TNFA )308A allele was associated with RF RHD [47, 48]. Of note, one study did not confirm the association in the Turkish population [49]. Thus, variants of TNFA may be one of the predisposing risk factors for RF RHD, which is likely to act in synergy with other factors, both genetic and environmental, in the development of the disease.

4 202 Rheumatic Fever and Rheumatic Heart Disease L. Guilherme et al. The molecular mechanism underlying the association of TNFA )308A G with susceptibility to RF RHD is not known. TNFA )308A was reported to be associated with high TNF-a production [50]. TNF increases the synthesis of proinflammatory cytokines and also leads to the activation of NFjB which subsequently activates the transcription of proinflammatory genes. Several lines of evidence indicate a key role for TNF-a in the immunopathogenesis of RF RHD as presented below in the pathogenesis mechanisms section. Several other candidate genes have been investigated with negative or positive findings, including angiotensinconverting enzyme [51 53], Fcgamma RIIA and Fcgamma RIIIB [54] and TGF-b [55]. Further follow-up studies will be needed with much larger sample sizes to replicate the findings. Overall, the identification of the HLA-DR molecules associated with RF RHD led to the elucidation, in part, of the mechanism underlying the pathogenesis of the disease, which will be discussed in the pathogenesis mechanism section. Currently, only a few genes for RF RHD have been identified through candidate gene-association studies. Future studies will need to focus on immune response gene pathways to unravel the breaking of tolerance in these patients to tip them towards the development of autoimmunity. Pathogenic mechanisms Rheumatic fever or rheumatic heart disease is the most convincing example of molecular mimicry in human pathological autoimmunity, given the cross-reactions between streptococcal antigens and human tissue proteins, mainly heart tissue proteins, which follow throat infection by S. pyogenes in susceptible individuals. The S. pyogenes, or group A streptococci (GAS), cell wall consists of carbohydrates such as N-acetyl b D-glucosamine linked to a polymeric rhamnose backbone. Group A streptococci contain M, T and R surface proteins and lipoteichoic acid (LTA), all of which are involved in bacterial adherence to throat epithelial cells. The M protein, which extends from the cell wall, is composed of two polypeptide chains with approximately 450 amino acid residues, in an a-helical coiled-coil configuration. The amino-terminal (N-terminal) portion presents antigenic variations but is highly homologous, with the exception of the first 11 amino acid residues that define the different serotypes [56], of which 200 have been identified to date. The M protein is the most important antigenic structure of the bacteria and shares structural homology with a-helical coiled-coil human proteins like cardiac myosin, tropomyosin, keratin, laminin, vimentin and several valvular proteins [reviewed 57]. During the acute phase of the throat infection, inflammatory acute phase proteins, such as MBL, and the cytokines IL-1, IL-6 and TNF-a should be produced to eliminate the bacteria. All these proteins are genetically controlled and in RF RHD patients, alterations or mutations can lead to differential expression and or secretion, consequently causing damage. MBL binds to the N-acetyl b D-glucosamine in the streptococcal cell wall, activating the complement-lectin pathway that induces the clearance of the bacteria. Some mutations in the gene that codes for MBL production present in RF and RHD patients probably interfere in the clearance of S. pyogenes (R. Ramasawmy, G. Spina, K. C. Faé, A.C. Pereira, R. Nisihara, I. Messias Reason, M. Grinberg, F. Tarasoutchi, J. Kalil, L. Guilherme, unpublished observations). In addition, RF RHD patients presented increased levels of TNF-a in the plasma [58 60] and it was demonstrated that stimulation of peripheral blood mononuclear cells from children with ARF with streptococcal antigen produced higher levels of TNF-a when compared with controls [61]. IL-1 is also reported to increase during active rheumatic carditis [62]. Molecular mimicry and autoimmune reactions Molecular mimicry is a mechanism by which host and pathogen antigens that exhibit some degree of homology are recognized through cross-reactivity by both T and B lymphocytes. The antibodies secreted by B cells recognize the conformations of proteins and other antigen molecules. T cells recognize peptide fragments combined with MHC molecules. The pathogenesis of RF RHD seems to result from an overt immune response involving either humoral or cellular reaction or both, triggered by group-a streptococci infection. The concept of an involvement of autoimmune reactions in the pathogenesis of RF was introduced only in the 1960s by Kaplan who demonstrated that antibodies against GAS reacted with human heart preparations [63, 64]. Subsequent studies conducted by Zabriskie et al. gave support to the hypothesis that RF has an autoimmune origin by describing the presence of antibodies that were cross-reactive with streptococcal membrane antigens in acute RF sera [65, 66]. Goldstein and colleagues showed that antibodies to the N-acetylglucosamine carbohydrate cross-reacted with glycoproteins present in the heart valves that contain N-acetylglucosamine [67]. Cunningham s group has shown that human monoclonal antibodies also react with N-acetylglucosamine, cardiac myosin and laminin. They have also demonstrated the in vitro cytotoxic activity of human and murine monoclonal antibodies [57]. Anti-M protein antibodies were shown to cross-react with vimentin and cardiac myosin, suggesting that these proteins were the target autoantigens recognized in the heart [68 72]. Using anti-myosin antibodies purified by affinity from ARF patient sera, the same group identified cross-reactive

5 L. Guilherme et al. Rheumatic Fever and Rheumatic Heart Disease 203 epitopes on myosin and the M5 M6 proteins [73]. In addition, they demonstrated the potential role of these cross-reactive antibodies in the development of RHD, by showing that they are able to bind to the endothelial surface, which may lead to inflammation, cellular infiltration and valve scarring [74]. The upregulation of the adhesion molecule VCAM-1 after binding of cross-reactive antibodies to the valvular endothelium facilitates cellular infiltration [75] (Fig. 2). The first evidence of CD4 + T-cell involvement in RHD lesions was described by Raizada et al. [76], while their role in the development of heart tissue lesions in RHD was described later by us [77]. We succeeded in growing in vitro these heart tissue infiltrating T cells and through a molecular analysis we demonstrated the molecular mimicry between b-haemolytic streptococci and heart tissue proteins. By generating T-cell clones from heart lesions of four severe RHD patients, we demonstrated for the first time the ability of 7.5% of these cells to simultaneously recognize M protein peptides and heart tissue-derived proteins. Three M5 regions (residues 1 25, and ) were cross-reactive with several heart protein fractions, mainly those derived from valvular tissue with molecular masses of , and kda [77](Fig. 2). The frequencies of autoreactive T cells were higher in RHD patients in acute phase of the disease (67%) when compared with RHD patients in chronic phase (around 25%) [78]. Peptides included in the three immunodominant M5 regions described above and heart tissue protein fractions were also recognized by peripheral T cells from RF RHD patients [79]. The M5(81 96) epitope was preferentially recognized by patients with severe RHD that expressed HLA-DR7 + DR53 + [79]. These data suggest a role for the HLA class II molecules DR7 and DR53 in presenting the streptococcal immunodominant peptide to the TCR. In addition, show the significance of the propensity of DR7 and or DR53 to cause severe RHD in Brazilian patients and probably other populations in which this class II allele is associated with RHD and with the development of valvular lesions. Yoshinaga et al. [80], isolated T cells from heart valves and compared the reactivity of PHA-stimulated T-cell lines derived from heart-valve specimens and peripheral blood lymphocytes of RF patients and showed that, even though these cells recognized cell wall and membrane streptococcal antigens, they failed to react with the M protein, myosin, or other mammalian cytoskeletal proteins. The absence of reactivity against M protein and self-antigens probably was a result of low frequencies of specific T cells. Myosin M5 protein cross-reactive T-cell epitopes were also investigated in mice immunized with intact cardiac myosin. Lymph node T cells cross-reacted with overlapping M5 peptides and seven regions, termed NT4 5 6, Table 2 M protein peptides recognized by human and murine T cells. Amino acid residues M5(1 35) AVTRGTINDPQRAKEALDKYELENHDLKTKNEGLK a M5(1 25) TVTRGTISDPQRAKEALDKYELENH b NT5(59 76) KKEHEAENDKLKQQRDTL a NT6(72 89) QRDTLSTQKETLEREVQN a M5(81 96) DKLKQQRDTLSTQKET b M5(83 103) LKQQRDTLSTQKETLEREVQN b M5(91 103) STQKETLEREVQN b NT5 NT6 myosin cross-reactive peptides described by Cunningham et al. [81], and human valvular tissue proteins cross-reactive M5(1 25), M(81 96), M5(81 103), M5(91 103) peptides described by Guilherme et al. [77]; M5(1 35) peptide described by Robinson et al. [82]. Bold typed and underlined regions correspond to the identical residues among the different peptides. a Murine lymph node T cells. b Human heart infiltrating T-cell clones. B1B2, B2, B2B3A, and B3A, were predominantly recognized [81]. NT5 6 and B1B2 B2 aligned with the M5 regions previously identified by our group, namely M5(81 103) and M5( ) respectively. Robinson et al. [82], obtained lymph node T-cell clones from mice immunized with recombinant M5 protein, and the M5(1 35) peptide aligned with the M5(1 25) region recognized by human heart-infiltrating T-cell clones (Table 2). Cardiac myosin is one of the major autoantigens involved in rheumatic heart lesions [57, 71]. Recently, we showed a very high frequency (63.2%) of heart tissue infiltrating T-cell clones recognizing the myosin ß chain, the LMM fragment. Thirty-four per cent of T-cell clones exhibited cross-reactivity with different patterns such as, (1) myosin and valve-derived proteins; (2) myosin and streptococcal M5 peptides; and (3) myosin, valve-derived proteins and M5 peptides [83]. The reactivity against LMM peptides of heart tissue infiltrating T cells may be attributable to the stimulation of these cells initially by the a-helical coiled-coil streptococcal M protein. The myosin cross-reactivity with several other valvular proteins may occur first through mimicry [77, 84] and eventually by an epitope spreading mechanism [85] in which the evolution of an autoimmune pathology may easily obliterate any evidence of the initial target antigen, such as streptococcal M protein and cross-reactive self-antigens. Cross-reactive peripheral T-cell clones from a RHD patient were responsive to group A recombinant M6 protein, cardiac myosin, tropomyosin, and laminin, a valve protein [84]. The authors showed that the cross-reactive response was MHC class I- and II-restricted for T-cell clones that were CD4 + and CD8 + respectively. These cells recognized streptococcal M6 epitopes on the B region, and S2 and LMM regions of human cardiac myosin [84]. Both peripheral and rheumatic heart lesion-derived T-cell clone studies demonstrate mimicry at the T-cell

6 204 Rheumatic Fever and Rheumatic Heart Disease L. Guilherme et al. Patient # 1 Vβ13-Cβ Vβ13-Cβ 10 PBMC Polyclonal profile 10 HEART- LESIONS Infiltrating T- cell line Oligoclonal profile of T cell expansion bearing TCR-Vß13 Jß2.7 Jß2.7 Figure 1 CDR3 size patterns of a heart tissue expanded T-cell population. Top left, patient no. 1, Vß13 family found in peripheral blood mononuclear cells with a polyclonal pattern. Top right, Vß13 family found in the mitral valve-derived T-cell line as an oligoclonal expansion that combines with some Jß segments. Vß13Jß2S7 an oligoclonal expansion that corresponds to 61.8% of the expanded T cells bearing the TCRVß13 (Table 3). level between streptococcal M protein and human cardiac myosin epitopes and confirm our previous work reported for T-cell mimicry in the valve. We observed that IFN-c, TNF-a (inflammatory cytokines) and IL-10 (regulatory cytokine) positive cells were consistently predominant in both myocardium and valvular tissue whereas IL-4 (regulatory cytokine) was scarce in the valves. The predominance of inflammatory cytokines in the heart lesions confirms that RHD is mediated by inflammatory immune responses. In addition, the significantly lower IL-4 expression in the valvular tissue may contribute to the progression of RHD leading to permanent damage in the valves [86] (Fig. 2). The heart tissue infiltrating T-cell repertoire is probably composed by migrating peripheral primed T cells. We assessed the clonality of T cells by analysing the Vß and Jß genes of the TCR in both peripheral blood and intralesional T-cell lines obtained from heart tissue from several RHD patients. The relative frequency of the 22 Vß families in both peripheral and intralesional T-cell lines showed no particular expansion of any Vß family. The analysis of TCR Vß and Jß rearrangement showed the clonal presence of expanded families in the heart tissue and a polyclonal distribution in the peripheral blood [88]. Here, we present some examples of these patterns in Fig. 1 and Table 3. The hypothesis that streptococcal primed T cells migrate from Table 3 Relative frequencies and CDR3 patterns of T cells from the peripheral blood and mitral valve-infiltrating T-cell lines. Patient no. Origin of T cells subsets Vb family CDR3 pattern Jb family CDR3 pattern 1 PBMC Vb % Polyclonal ND Mi V 87.2% CD % CD8 + Vb % Oligoclonal Jb % Jb % Jb % Jb % All oligoclonal 2 PBMC Vb 1 2.7% Polyclonal ND Mi V 91.0% CD % CD8 + Vb 1 7.6% Oligoclonal Jb % Jb % Jb % All oligoclonal 3 PBMC Vb 5 1.7% Polyclonal ND Mi V 49.4% CD % CD8 + Vb 5 5.3% Oligoclonal Jb % Jb % All oligoclonal PBMC, peripheral blood mononuclear cell. CDR3 patterns were analysed by the Immunoscope approach [87]. Bold typed, means major expansions. ND, not done.

7 L. Guilherme et al. Rheumatic Fever and Rheumatic Heart Disease 205 Valvular tissue CD4 + T cells Cross reactive antibodies Mononuclear cells Streptococcal/self antigens cross reactive antibodies facilitate heart- tissue T cells infiltration CD4 + T cells recognize streptococcal antigens and heart-tissue proteins by molecular mimicry Inflammatory cytokines (TNF-α IFN-γ) are produced by mononuclear cells Surgical specimen of acute RF showing small vegetations on the line of closure of the mitral valve (arrows). Macroscopic photo done by Aiello, VD Heart Institute (InCor). Few mononuclear cells produce IL-4 (regulatory cytokine) leading to the progression and maintenance of valvular lesions. Figure 2 Major events triggering rheumatic valvular lesions in RHD. Heart tissue cross-reactive antibodies bind to the surface of the valve endothelial and facilitate mainly CD4 + T-cell infiltration [75 77]. Streptococcal primed T cells trigger an autoimmune reaction and produce inflammatory cytokines. Low numbers of regulatory IL4 + cells are found in the valvular tissue [88]. The permanent ongoing inflammation leads to the valvular lesions such as the small vegetations. the periphery to the heart was confirmed by the identification of some M protein heart tissue cross-reactive intralesional CD4 + T-cell clones. As an example, an intralesional T-cell clone that recognized two cardiac myosin peptides (LMM39 and LMM41, amino acid residues and respectively) and a mitral valve-derived protein with a molecular mass of kda and isoelectric point of 6.76 displayed the TCR-Vß13Jß2S7 [80]. Taken together, these results demonstrate that T-cell populations are expanded in heart lesions, driven by selfantigen recognition, and probably contribute to the maintenance and progression of tissue damage. Concluding remarks All the findings described in the last 50 years about the immune response leading to RF and RHD have led to a significant understanding of the pathogenesis of RF and RHD. The association of HLA class II alleles, considered as genetic markers for RF RHD, in fact pointed out HLA molecules that preferentially present streptococcal and selfantigens to the TCR. The gene TNFA, located on the same chromosome as the HLA genes, carrying a mutation in the promoter region leading to the increased production of TNF-a, an inflammatory cytokine, is associated with the disease. Other genes related to immune regulation are now being investigated and will certainly contribute to the definition of new genetic markers and an understanding of how these genes act in the complex network of autoimmune reactions that occur in RF RHD. Both B and T cells play important role in the development of RF. In Fig. 2, we summarize the major events leading to RHD valve lesions, such as the small vegetations shown on the left side of the picture. Streptococcal and host antigen cross-reactive antibodies facilitate heart tissue infiltration by T lymphocytes. CD4 + T cells are the major effectors of the heart tissue, leading to RHD lesions. Inflammatory cytokines such as, TNF-a and IFNc are the mediators of valve lesions and the presence of few mononuclear cells producing IL-4, a regulatory cytokine, leads to the progression and maintenance of valve lesions in RHD patients. Acknowledgment We acknowledge Dr Vera Demarchi Aiello from the Heart Institute (InCor) for providing us with the macroscopic photo of ARF valvulitis. References 1 Dajani AS, Ayoub E, Bierman FZ et al. Guidelines for the diagnosis of Rheumatic Fever: Jones criteria, 1992 uptade. JAMA 1992;268: Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal diseases. Lancet Infect Dis 2005;5: Wilson MG, Schweitzer MD. Pattern of hereditary susceptibility in rheumatic fever. Circulation 1954;10: Gray FG, Quinn RW, Quinn JP. A long term survey of rheumatic and non-rheumatic families; with particular reference to environment and heredity. Am J Med 1952;13: Uchida IA. Possible genetic factors in the etiology of rheumatic fever. Am J Hum Genet 1953;5: Stevenson AC, Cheeseman EA. Heredity and rheumatic fever; a study of 462 families ascertained by an affected child and 51 families ascertained by an affected mother. Ann Eugen 1953;17:

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