Background
Chagas disease is an anthroponosis caused by the protozoa parasite
Trypanosoma cruzi and is a leading cause of chronic cardiomyopathy in Latin America [
1]. In its natural condition, the disease is transmitted by infected feces from triatomines. Recently, contaminated food has emerged as an important transmission source, and, to a minor extent, infections happen by congenital transmission and blood transfusion [
1,
2]. The disease has spread from its original boundaries in South America through international migration and has become a new concern for blood donation worldwide [
3].
About 30% of the infection progresses to cardiomyopathy and 8–10% progress to mega syndromes, mainly in esophagus and colon, culminating in organ hypertrophy and dilatation [
1]. Chronic Chagas disease cardiomyopathy (CCC) is a life-threatening disease that leads to direct complications in the cardiovascular system, such as conduction disorders and heart failure, being the most important cause of death in infected individuals [
4]. CCC is characterized by a multi-focal inflammatory infiltrate within the myocardium, mainly characterized by infiltrating CD8
+ and CD4
+ T cells, macrophages, and progressive interstitial fibrosis [
5,
6]. The disease’s progression is highly variable, and individuals may stay in an indeterminate clinical form for decades until the first clinical symptoms emerge. Chronic inflammation results in a tissue remodeling that leads to loss of function, and in several cases to the need for heart transplantation [
7].
The mechanisms underlying the progression of CCC are poorly understood and probably multifactorial. The persistence of
T. cruzi in tissue provides the antigenic load that sustains local inflammation, although the presence of the parasite in myocardium is scarce [
5,
8,
9]. CD8
+ T cells are essential for parasite control in murine models by promoting cell death of infected cardiomyocytes through main-histocompatibility complex class-1 (MHC-I) antigen presentation [
10]. In contrast, the role of CD4
+ T cells has been more obscure in the pathogenesis of CCC.
Trypanosoma cruzi-specific IFN-γ
+CD4
+ T cells are increased in CCC patients after antigenic recall when compared to patients in the indeterminate form of Chagas disease [
11,
12]. In addition, the IFN-γ-induced chemokine receptors CCR5 and CXCR3 are upregulated in CD4
+ T cells during established cardiomyopathy suggesting the participation of a type-1 T helper-mediated immunity (Th
1) in the disease progression [
13,
14]. Along with the higher proportion of Th
1 cells in CCC patients, a lower frequency of multifunctional CD4
+ T cells in the circulation and a higher frequency of senescent and/or exhausted helper T cells have been associated with the progression of the heart disease [
15,
16]. Murine models shed light on mechanistic processes in which CD4
+ T cells may regulate or promote myocarditis [
17,
18], but whether the CD4
+ T cell accumulation in myocardium is a cause or a consequence of progressive cardiomyopathy, is still under investigation.
The complex network of CD4
+ T cell phenotypes during CCC suggests a shift towards inflammation while immunoregulatory mechanisms seem to be progressively damped [
8,
19,
20]. In this sense, the role of specific T helper phenotypes in either promoting or regulating tissue damage deserves further exploration. Also, the lack of reliable markers that correlate with the progression of CCC urges the need for biomarkers that enable the prediction of clinical progression [
21].
Here, we used high dimensional flow cytometry to phenotype CD4+ T cells from patients in different stages of chronic Chagas disease compared to healthy individuals. We found phenotypic signatures using unsupervised data analysis, which are associated with the immunopathological processes of CCC progression.
Discussion
We used high dimensional flow cytometry to assess the heterogeneity of CD4
+ T cells from patients in various stages of Chagas disease. Clinical classification of patients is challenging due to the heterogeneous clinical presentation, and the difficulty to detect the very early myocardial injury. In this study, patients with Chagas disease were segregated in those who have no signs of cardiomyopathy (group A) from those with mild chronic cardiomyopathy (group B1) and patients with established chronic cardiomyopathy (group B2-C-D) [
1,
22].
Unsupervised clustering revealed an increase of CD69
+ cells among T
N (MC1) and memory (MC4 and MC5) CD4
+ T cells in PBMC from patients with Chagas disease. CD69 is rapidly upregulated in CD4
+ T cells after activation and is involved in their retention in lymph nodes due to CD69-mediated downregulation of sphingosine-1 phosphate receptor (S1PR1), which leads to very low proportions of CD69
+ T cells in blood under homeostatic conditions [
26,
27]. CD69 is constitutively co-expressed with CD103 on mucosal and skin resident memory T cells (T
RM) [
28]. Indeed, CD69 gene expression is upregulated in the heart from CCC patients compared to healthy controls [
29,
30]. The absence of T
RM in the circulation suggests these cells are not prone to recirculate [
28]. Thus, the expansion of CD69
+CD4
+ T cells during Chagas disease is probably not due to recirculation of T
RM. Furthermore, previous data have described an antigen-experienced population, named stem cell memory T cells (T
SCM), that displays strong multipotency and can give rise to all memory subsets [
30,
31] and is characterized by expressing a naïve phenotype [
32]. The hallmark of T
SCM is the expression of CD95, which is not observed in MC1 (Fig.
3D).
Although the expression of CD69 on naïve CD4
+ T cells has been published in some pathological conditions, such as Graves’ disease and autoimmune thyroiditis [
33], no reports were found in Chagas disease. Patients with the indeterminate clinical form displayed a higher frequency of CD69
+CD4
+ γδ T cells as compared to healthy individuals and CCC, either ex vivo or after stimulation with trypomastigote protein fraction [
34]. In addition, CD69 expression was increased among CD25
highCD4
+ T cells from both indeterminate and CCC compared to healthy controls, either ex vivo or after stimulation with epimastigote antigen [
35]. In our study, a consistent increase in the frequencies of CD69
+CD4
+ T cells among all T cell subsets was observed during Chagas disease. Notably, CD69 was the only marker analyzed able to distinguish B1 from A and B2-C-D.
The increased frequency of CD69
+CD4
+ T cells suggests a constant activation by remaining parasites or antigens that persist into the Chagas disease’s chronic phase [
9]. Moreover, under certain conditions, CD69 expression may also be induced by IL-6, TNF, and IL-2 in naïve and memory T cells [
36]. In this sense, either antigenic stimulation or chronic release of pro-inflammatory cytokines could induce and sustain an activated status among CD4
+ T cells in patients with Chagas disease.
It has been suggested that pro-inflammatory mechanisms during cardiomyopathy are partially modulated by regulatory mechanisms that involve regulatory T cells [
11,
19,
20,
37‐
39]. Here we found a decreased frequency of regulatory T cells, primarily due to lower numbers of CD39-expressing T
REG, which was more pronounced among patients in the B1 group. Among regulatory T cells, those expressing CD39 are apparently more stable in inflammatory conditions and more efficient in immunosuppressive function [
40,
41]. Furthermore, the suppressive function of CD39
+ T
REG has been studied in different diseases. Decreased frequencies of CD39
+ T
REG are associated with inflammatory diseases such as multiple sclerosis, while increased frequencies are found in human colorectal cancer allowing cell proliferation [
42,
43]. Regulatory T cells from CCC patients were previously reported as not being able to suppress PBMC proliferation [
44]. Our findings also suggest that chronic Chagas disease progresses with loss of regulatory function and an increased pro-inflammatory signature in established cardiac disease. The contrasting results found by others [
35,
37] might be explained by the differences in the immunophenotyping strategy and the ex vivo approach versus the response induced by antigenic stimulation. In the present work, T
REG were analyzed ex vivo and were defined by the expression of FoxP3 and CD25.
Here, GrB
+PFN
+CD4
+ T cells were expanded in patients with Chagas disease compared to controls. Previous reports have shown an expansion of cytotoxic CD4
+ T cells during Chagas disease [
45‐
47]. CD4
+ T cells with high expression of cytotoxic granules may be linked to apoptosis induction in, but not limited to, antigen-presenting cells [
48]. Importantly, MHC-II is upregulated in heart endothelial cells from CCC patients, suggesting a potential role of cytotoxic CD4
+ T cells in directly induced death of these cells [
49]. Several different phenotypes of cytotoxic CD4
+ T cells have been described and are considered to add protection during viral infections and cancer [
48,
50]. CD45RA
+CCR7
−CX3CR1
+CD4
+ T cells expressing cytotoxic granules accumulate with multiple infections by Dengue virus [
50]. Compatible with this phenotype, we described the metacluster 9 that was expanded in patients with Chagas disease, express a T
EFF phenotype (CD45RO
−CCR7
−) and co-express the cytotoxic granules GrB and PFN, thus suggesting that these cells exhibit the same function.
CD4
+ T cells also produce several cytokines during chronic Chagas disease [
51,
52]. Increased responses were observed in patients with Chagas disease, with the B1 group displaying the higher proportion of responders and the higher frequencies of IFN-γ
+ activated CD4
+ T cells, mainly among T
EM. Multifunctional CD4
+ T cells were observed to a lesser extent, and most of the responding cells co-express IFN-γ and TNF while lacking the expression of IL-2. Others have found a predominance of CD4
+ T cells producing a single cytokine (IFN-γ, IL-2, or TNF) when using
T. cruzi amastigote and trypomastigotes antigens [
46,
53]. A higher frequency of IFN-γ
+IL-2
−CD4
+ T cells, a phenotype associated with low parasite load and proliferation, has also been shown in patients in the indeterminate form compared to CCC [
53].
Noteworthy, the antigen-specific CD4
+ T cells producing cytokines were mostly represented by metaclusters 7 and 25, co-expressing GrB and PFN. Indeed, a GrB
+PFN
+IFN-γ
+CD4
+ T cell population was expanded in Chagas’ patients [
46], suggesting that this population displays a role in CCC. Notably, host cytotoxic granules were shown to induce the direct killing of
T. cruzi [
54]. Altogether, these findings suggest a potential role of cytotoxic CD4
+ T cells in chronic cardiomyopathy in Chagas disease, which may be inducing cell death of
T. cruzi infected cardiomyocytes and endothelial cells, and producing cytokines that help effector functions. Whether their role is protective or detrimental in promoting cardiomyopathy is still unknown.
Finally, B1 patients displayed a unique signature of CD4
+ T cell populations during Chagas disease. The expansion of cytokine-producing CD4
+ T cells with cytotoxic activity, contemporaneously with contraction of CD39
+ T
REG, represented by mesocluster 6, suggests an imbalance of pro-inflammatory/regulatory responses. This imbalance may be associated with the cardiomyopathy progression initially observed in B1 patients. Furthermore, B1 has a unique expression of CD69 compared to other forms of Chagas disease, more pronounced among naïve T cells. Thus, the T cell signature observed in mild cardiomyopathy is expected due to more active myocarditis and tissue remodeling than patients with the indeterminate form. Indeed, IFN-γ-related chemokines are increased in B1 than indeterminate patients [
14]. Moreover, the IFN-γ effector signature observed in B1 patients may favor the control of parasite load. Considering that persistent parasitemia may contribute to cardiomyopathy progression, we hypothesize that persistent antigenic stimulation slowly leads to the accumulation of certain effector T cells that promote lesion in the heart as observed in B2-C-D.
This is the first study using high-dimensional flow cytometry in the context of Chagas disease. The cell signatures described here revealed an imbalance of the inflammatory/regulatory response which is more pronounced during mild cardiomyopathy. Our results provide new insights on the pathogenesis and development of different clinical forms of Chagas disease. Additionally, we propose the combination of the markers here described as a supplementary tool for the clinical management of patients.
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