Introduction

According to the World Health Organization (http://www.who.int/chagas/en/) and the Center for Disease Control and Prevention (https://www.cdc.gov/parasites/chagas/), 8 million people suffer from Chagas disease worldwide, mainly in Latin America. This disorder is caused by the infection with the protozoan parasite Trypanosoma cruzi [1, 2]. Chagas disease has two clearly identified stages, i.e. an early acute phase and a chronic phase, and approximately 30% of infected individuals develop cardiac and/or digestive complications [1]. It is believed that host genetic factors could be implicated in the development of these complications, as it has been observed in other infectious diseases [3]. During the last years, several studies have been carried out in Chagas endemic populations, reporting evidences of a genetic component influencing the susceptibility to infection by T. cruzi and/or chronic Chagas cardiomyopathy [4].

The innate immune response to T. cruzi starts immediately after infection by controlling the replication and spread of the parasite in the host tissues. In this phase, several cytokines including interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and reactive nitrogen species, regulate parasite invasion. Subsequently, an adaptive response characterized by a strong and persistent Th1 response dependent of IL-12 takes place, which facilitate the parasite clearing in the host [5]. Some studies suggest that an uncontrolled inflammatory response may originate chronic complications [5–7]. However, other studies point to the possible role of parasite persistence to originate such complications [7, 8]. From a genetic point of view, different genes of the inflammatory immune response have shown statistically significant association with a differential risk to infection and to the development of chronic cardiomyopathy. Therefore, it seems that the inflammatory immune response is the clue for controlling infection in early stages and to avoid the appearance of more severe symptoms. Altogether, the evidence indicates that parasite persistence is fundamental for the onset of chronic Chagas cardiomyopathy.

The tyrosine kinase 2 (TYK2) gene encodes a member of the Janus kinases (JAK) family that is essential for signal transduction of type I interferons, and for the interleukins (IL) IL-6, IL-10, IL-12, and IL-13. Knockout mice for Tyk2 displayed reduced responsiveness to type I interferons, lowered production of IFN-γ in spleen cells after treatment with IL-12 and IL-18, altered production of NO, increased susceptibility to viral infection, and altered differentiation into Th1 cells from naïve helper T cells [9, 10]. Moreover, it has been described in a previous report that the minor allele of some genetic variants mapping to this locus were able to reduce the function of the gene in mediating cytokine signaling in vitro and in a humanized mouse model [11]. In humans, patients with genetic mutations on TYK2 exhibited multiple deleterious effects in the immune response, including impaired responses to IL-12 and type I interferons, increased susceptibility to microorganisms (virus, fungi and mycobacteria), and an imbalanced Th1/Th2 differentiation [12, 13]. Therefore, and based on all the above, we decided to perform a genetic analysis of the TYK2 gene in order to evaluate its possible influence in the susceptibility to infection by T. cruzi and/or the development of Chagas cardiomyopathy.

Materials and methods

Results

The three TYK2 SNPs were in HWE in all the comparisons performed (P>0.01), and the genotyping success rate was over 95% for all analyzed genetic variants. In all cases, r² values between each pair of SNPs were < 0.25, indicating independency amongst them.

In order to evaluate the association between TYK2 gene variants and susceptibility to T. cruzi infection, the allelic frequencies of seronegative and seropositive individuals were compared (Table 1). For all tested SNPs, no statistically significant differences were observed in this comparison, thus suggesting that they do not influence the risk of infection by T. cruzi.

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Table 1. Genotype and allele distribution for TYK2 polymorphisms in seropositive and seronegative individuals.

https://doi.org/10.1371/journal.pone.0190591.t001

On the other hand, we evaluated the possible association between TYK2 genetic variants and chronic Chagas cardiomyopathy by comparing the allelic frequencies of the TYK2 SNPs between chronic Chagas cardiomyopathy individuals and seropositive asymptomatic individuals. However, no statistically significant differences were observed for any of the analyzed genetic variants (Table 2).

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Table 2. Genotype and allele distribution for TYK2 polymorphisms in chronic Chagas cardiomyopathy (CCC) and asymptomatic individuals.

https://doi.org/10.1371/journal.pone.0190591.t002

Discussion

To our knowledge, this is the first study aimed to examine the role of functional genetic variants on TYK2 in a parasitic disease. Specifically, we have analyzed common and low-frequency variants of the gene and their possible implication in the susceptibility to Chagas disease and its progression to Chagas cardiomyopathy. Our results suggest that TYK2 does not seem to play an important role neither in the predisposition nor in the progression of an infectious condition like Chagas disease.

The influence of TYK2 genetic variants have been previously assessed in other infectious diseases, such as tuberculosis [22] and in the human immunodeficiency virus (HIV) [18]. However, no significant associations have been described so far, which is consistent with our findings, even though the sample size of our study population provided an 80% statistical power to detect an OR = 2. Interestingly, the role of both rs34536443 and rs12720356 has been exhaustively analyzed by Dendrou and colleagues, in autoimmune diseases, other immune-mediated conditions [11]. The authors performed a meta-analysis including ten autoimmune diseases with further molecular, cellular, in vivo and structural follow-up studies. Although the rs12720356 variant showed inconsistent results among the different diseases, rs34536443*C was established as the lead independent signal conferring protection against all studied diseases, being able to impair the signaling of type I IFN, IL-12, and IL-23. However, this reduction on cytokine activity was not associated with immunodeficiency. Furthermore, individuals having the risk allele did not show an increase in hospitalization due to mycobacterial, bacterial, viral, or fungal infections. All this evidences indicate that rs34536443*C may allow a sufficient TYK2-mediated cytokine signaling to prevent immunodeficiency [11]. In light of this data, it is not surprising that a similar scenario may be plausible in infectious diseases, where individuals with this genotype may have a minimal signaling activity that helps with the immune response against the infection and, consequently, no differences in the outcome of the disease are related to these particular SNPs.

On the other hand, the rs2304256 represents a non-synonymous change (Val362Phe) within the FERM domain of the encoded protein which has been implicated in the interaction with transmembrane proteins such as cytokine receptors, binding the kinase domain and positively regulating its catalytic activity [23]. According to prediction tools [24, 25], the Val362Phe amino acid change is likely benign, thus indicating that it favors the protein activity.

In conclusion, given our results and previous evidences, the TYK2 functional variants analyzed in this study are not likely important players in the risk of T. cruzi infection. However, we cannot rule out the possibility that a potential lack of sufficient statistical power may be limiting our results. In this work we show that none of the genetic variants are associated to Chagas disease susceptibility and/or progression to Chagas cardiomyopathy on infected individuals. Further studies in different populations may be necessary to confirm our findings and to serve as a basis for a better understanding of the genetic risk factors of Chagas disease.

Acknowledgments

We thank Sonia García and Gema Robledo (from the Instituto de Parasitología y Biomedicina ‘López-Neyra’, CSIC, Spain) for their excellent technical assistance and all of the patients and healthy controls for kindly providing their essential collaboration.