Genetic susceptibility to Chagas disease cardiomyopathy: involvement of several genes of the innate immunity and chemokine-dependent migration pathways

Chagas disease (American trypanosomiasis) is caused by the protozoan Trypanosoma cruzi and transmitted by the reduviid bug. It occurs exclusively in the Americas, particularly in poor, rural areas of Mexico, Central America, and South America. The disease remains endemic in Latine America where the vector-based transmission is still active in some countries. Imported disease is increasingly recognized as an emerging problem in the USA and Europe due to immigration from Latin America. It is estimated that as many as 8–9 million people have Chagas disease. Approximately, 40 million people are currently at risk of infection [1]. Decades after acute infection, approximately 30% of infected individuals develop Chronic Chagas cardiomyopathy (CCC), one of the most important consequence of T. cruzi infection. CCC is an inflammatory dilated cardiomyopathy, with a potentially fatal outcome. 5 to 10% of infected individuals develop digestive disease. The remaining two-thirds of infected individuals remain asymptomatic (ASY) and free from heart disorders for life [2]. 20,000 deaths attributable to Chagas disease occur annually, typically due to CCC [3]. Heart failure due to CCC has a worse prognosis with 50% shorter survival when compared to other cardiomyopathies of different etiologies [4, 5].

The dynamics of the immune response to T. cruzi is that of a persistent infection with an obligatory intracellular parasite. During acute T. cruzi infection, T. cruzi pathogen-associated molecular patterns (PAMPs) trigger innate immunity in multiple cell types [6], which release proinflammatory cytokines and chemokines, such as IL-1, IL-6, IL-12, IL-18, TNF-α, CCL2, CCL5, and CXCL9 activating and mobilizing migration of cascades of inflammatory cells [7, 8]. Antigen-presenting cells subsequently elicit a strong T cell and antibody response against T. cruzi, where IL-12 and IL-18 drive the differentiation of IFN-γ-producing T. cruzi–specific Th1 T cells which migrate to sites of T. cruzi-induced inflammation, including the myocardium, in response to locally produced chemokines [9, 10]. Th1 T cell and antibody responses lead to control but not complete elimination of tissue and blood parasitism, establishing a low-grade chronic persistent infection by T. cruzi. As a result of persistent infection, both CCC and ASY chronic Chagas disease patients show a skewed Th1-type immune response [11, 12], but those who develop Chagas cardiomyopathy display a particularly strong Th1-type immune response with increased numbers of IFN-γ-producing T cells in peripheral blood mononuclear cells (PBMC) [13] as well as plasma TNF-α in comparison with uninfected or ASY patients [14]. PBMC of CCC patients also display increased levels of IFN-γ- or TNF-α producing CCR5/CXCR3+ CD4+ T cells [15, 16]. In addition, CCC patients display a reduced number of CD4⁺CD25^highIL-10⁺ and CD4⁺CD25^highFoxP3⁺ regulatory T cells in their peripheral blood as compared to patients in the ASY form of Chagas disease, suggesting such cells may play a role in the control of the intensity of inflammation in chronic Chagas disease [15, 17]. Furthermore, PBMC from CCC patients displayed increased numbers of CD4⁺CD25^highFoxP3⁺CTLA-4⁺ T cells, and decreased numbers of as compared to ASY patients. These reports suggest that a smaller CD4⁺FoxP3+/CD25⁺ Treg compartment with deficient suppressive activity exists in CCC patients, leading to uncontrolled production of Th1 cytokines [18]. Circulating CD4⁺IL-17⁺ T cells appear in low frequency in PBMC from CCC patients as compared with ASY patients and non-infected individuals [18, 19]. On the whole, these results suggest that proinflammatory cells and cytokines are markers associated with progression to CCC, whereas the production of IL-10, IL-17 and increased numbers of regulatory T cells are markers of protection from CCC development, indicating that failure to regulate Th1 responses may be the underlying immune defect of patients who progress to CCC.

The exacerbated Th1 response observed in the PBMC of CCC patients is reflected on the Th1-rich myocardial inflammatory infiltrate, with mononuclear cells predominantly producing IFN-γ and TNF-α, with lower production of IL-4, IL-6, IL-7, and IL-15 [7, 20, 21]. It has recently been shown that CCL5+, CCXCL9+, CCR5+, CXCR3+ cells were abundant in CCC myocardium, and mRNA levels of the Th1-chemoattracting chemokines CXCL9, CXCL10, CCL2 (also known as MCP-1), CCL3, CCL4, CCL5; along with CCL17, CCL19, CCL21 and their receptors were also found to be upregulated in CCC heart tissue [12, 22]. Importantly, median expression of CCL5, a CCR5 ligand, was the highest among all chemokines tested (166-fold increase over control). Significantly, the intensity of the myocardial infiltrate was positively correlated with CXCL9 mRNA expression. Moreover, a single nucleotide polymorphism in the CXCL9 gene, associated with a reduced risk of developing severe CCC in a cohort study, was associated with reduced CXCL9 expression and intensity of myocarditis in CCC [22]. These results are consistent with a major role of locally produced Th1-chemoattractant chemokines in the accumulation of CXCR3/CCR5+ Th1 T cells in CCC heart tissue [23].

Familial aggregation of CCC has been described, suggesting that there might be a genetic component to disease susceptibility [24]. Several genes were associated to an increased risk to develop cardiomyopathy (HLA, MHC, TNF, IL1A, IL1B, IL1RN, IL10, IL12B, TIRAP, CCL2, BAT1, LTA, IKBL, CCR5, MIF, IFNG, CXCL9, CXCL10) [25‐50]. So far, up to 30 case control studies were done (see for review [51‐53]). These studies often led to inconclusive results that may be explained in different ways: a) the use of seronegative subjects as controls which are inadequate controls, since it is unknown whether they were exposed to the pathogen; b) the relatively small size of the study groups which affected the power (the probability) to detect an association; c) the number of tested SNPs; d) the highly heterogeneous genetic background of the study population due to admixture; e) the sex ratio known to exist has not been taken in consideration [54].

Among these susceptibility studies, putative implication of genes crucially involved in the innate immunity-such as the Toll like receptors (TLR) and some of its most relevant signalling molecules like TIRAP was searched for. Two studies on the TLR and TIRAP failed to identify disease associations with TLR 1,2, 5, 6 and 9; in one of the reports an association was found with a TLR4 SNP among Chilean chagasic patients [55], while in the second study – which enrolled nearly double the number of Brazilian Chagasic individuals - no association was found with TLR4, but instead with TIRAP S180L heterozygosity [41]. Chemokines are key players in controlling migration of specific cell types bearing their receptors to sites of tissue inflammation, and associations between CCR5 –involved in T cell and macrophage migration and CCL2 –involved in monocyte migration - with CCC were reported [42, 47, 48]. Both processes, TLR signaling and chemokine-mediated cell migration are of paramount importance in Chagas disease and are key to the pathogenesis of CCC. Here, we conducted a study focusing on TIRAP, CCL2 and CCL5. Thorough genetic analysis, testing multiple tag SNPs per gene and thus detecting any possible relevant genetic variants in a large Brazilian population and ASY subjects as controls we could have a sensitive assessment of the contribution of genetic variants in prognosis to CCC either confirming or finding additional associated SNPs in the mentioned genes. This can be considered a candidate gene replication study, performed with a larger cohort of Chagas patients and only comparing CCC to the asymptomatic seropositive (ASY) patient group. Significant associations were found for CCR5, CCL2, and TIRAP genes.

Fifteen Tag SNPs were genotyped successfully on our original cohort including ASY subjects (n = 118) and CCC patients (n = 315) (Table 4). The genotyping steps were done successfully for all the Tag SNPs. The genotype distribution of each SNP is summarized in Table 5. All the SNPs were in Hardy-Weinberg equilibrium on the ASY individuals considered as control subjects (p > 0,001) (Table 6).

Our data show beyond reasonable doubt that polymorphisms affecting key molecules involved in several immune parameters (innate immunity signal transduction and T cell/monocyte migration to inflammatory regions) play a role in genetic susceptibility to CCC development. However, the functional impact of these markers remains unknown. This also points out to the multigenic character of CCC, each polymorphism imparting a small contribution.

When all the genetic markers will be identified, we will be able to performed multivariate analyses using several genes (gene polymorphisms) as covariates. In order to perform this kind of analysis it is essential to enroll a study population including at least 1,500 and 2,000 cases and 1000 ASY controls. It will allow us to detect gene–gene interactions and additive or antagonist effects between the associated polymorphisms. A panel of markers will be defined to early detect individuals with a highest risk to develop chronic Chagas cardiomyopathy. It will provide information for pathogenesis as well as therapeutic targets. The identification of these marker sets may also have a combined prognostic value for disease progression at the individual patient level, allowing close follow up and early treatment of those carrying high-risk genetic signatures.