Specific activation of CD4–CD8– double‐negative T cells by Trypanosoma cruzi‐derived glycolipids induces a proinflammatory profile associated with cardiomyopathy in Chagas patients

Keywords: cytokines, inflammation, parasitic‐protozoan, T cells

INTRODUCTION

Infection of immunocompetent individuals by different pathogens may result in the establishment of an intense inflammatory response, leading to elimination of the infectious agent and cure of infection. Alternatively, it may trigger an immune response that is insufficient to control the pathogen fully, leading to the establishment of infection. In this case, either the individual will develop a severe disease and succumb to the infection, or the pathogen and the host will co‐exist. The latter is the most common outcome in parasitic infections, where host survival is essential for the parasite 1.

As an example, in chronic Chagas disease caused by infection with the protozoan parasite Trypanosoma cruzi, most patients (∼70%) remain in a mild/asymtomatic form (named indeterminate, IND), while approximately 30% develop severe cardiac (CARD) and/or digestive clinical forms, associated with cumulative tissue damage due to a lifelong illness 2. Cardiomyopathy is the most serious clinical outcome, responsible for at least 12 000 deaths every year and a global economic burden of more than 7 billion dollars/year 3. The mechanisms underlying this distinct clinical progression are not understood completely, but it is evident that parasite strain and tissue tropism, parasite load, time of infection and host immune responses are critical 4, 5. To date, there is no effective measure that can predict, prevent or revert disease progression in the chronic phase.

A predominantly anti‐inflammatory cytokine profile is often observed in IND patients, while an inflammatory profile is clearly associated with CARD 1, 5. Tumour necrosis factor (TNF) and interferon (IFN)‐γ expression have been associated with worse cardiac function 6 and with progression to severe forms of cardiomyopathy, respectively 7, 8. Conversely, interleukin (IL)‐10 and IL‐17 expression have been associated with a controlled response observed in IND and better cardiac function 9, 10, 11, 12. Monocytes and T cell subpopulations are major sources of the immunoregulatory cytokines produced in the chronic phase of Chagas disease 7, 13, 14, 15.

We demonstrated recently that a minor subpopulation of T lymphocytes that do not express the co‐receptors CD4 and CD8, named double‐negative T cells (DN T cells), are involved actively in the production of cytokines in patients with different clinical forms of Chagas disease 16. DN T cells can be divided further into two subpopulations, based on the expression of the alpha‐beta or gamma‐delta T cell receptors (TCR‐αβ and TCR‐γδ) 17. These cells recognize mainly antigens presented via non‐classic major histocompatibility complex (MHC) molecules belonging to the CD1 family 18. We have demonstrated previously that, although both subsets of DN T cells can produce inflammatory and modulatory cytokines, the DN γδ TCR subpopulation is a more pronounced source of IL‐10 in IND patients, while expressing predominantly proinflammatory cytokines in CARD 16. However, the antigenic components of T. cruzi that polarize DN T cell responses remain unknown.

The T. cruzi surface is covered by a complex array of different molecules that include glycosylphosphatidylinositol (GPI)‐anchored glycoconjugates such as GPI‐mucins, mucin‐associated surface proteins (MASP), trans‐sialidases (TS) and glycoinositol phospholipids (GIPLs). Those molecules may also be present in extracellular vesicles (EVs) shed by the parasites 19, 20. GPI‐mucins, GIPLs and EVs exhibit high structural diversity and antigenicity depending on the strain and/or life stage of the parasite 21, 22, 23, 24. These molecules, which act as pathogen‐associated molecular patterns (PAMPs), are Toll‐like receptor agonists (TLR‐2, ‐4 and ‐6), triggering cytokine and chemokine production, and promoting recruitment and activation of phagocytic cells 25, 26, 27.

The high diversity of T. cruzi antigens may have an implication in the immunopathogenic events not only in the acute but also in the chronic phase of Chagas disease. Processing and presentation of different parasite components provide a plethora of stimulus that leads to establishment of T cell‐mediated adaptive responses. Thus, different parasite components may induce functionally distinct T cell responses, which may be associated with distinct disease outcomes 28, 29. Given the importance of DN T cells as cytokine producers during chronic Chagas disease, and their clear association with distinct clinical forms, the aim of this study was to determine which components of T. cruzi trigger activation of these cells in human Chagas disease. Our results showed that glycolipid‐enriched fractions (GCL), but not protein (PRO)‐ or lipid (LIP)‐enriched fractions, led to activation of DN T cells, especially the TCR‐γδ subpopulation, from IND and CARD patients. Moreover, GCL induced preferential expression of IFN‐γ by activated DN TCR‐γδ from CARD but not IND, leading to the predominance of an inflammatory milieu in CARD, as measured by the increased IFN‐γ/IL‐10 ratio. Although GCL led to higher expression of TNF and IL‐10 by DN T cells from IND, the TNF/IL‐10 ratio remained unaltered. Thus, the induction of TNF was not sufficient to shift the response towards an inflammatory profile. These results identify GCL as the major T. cruzi component responsible for activation of DN T cells, associated predominantly with an inflammatory profile in CARD Chagas patients. These results suggest that modulating the response to GCL may be a valid strategy for controlling inflammation‐induced cardiomyopathy.

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PATIENTS, MATERIAL AND METHODS

Patients

A total of 18 volunteers were enrolled into in this study. Twelve volunteer patients had positive specific serology for T. cruzi and were within the chronic phase of the disease, with extremely well‐defined and polar clinical forms; six individuals displayed negative specific serological tests for Chagas disease and were included as a control group. Patients were from Chagas disease endemic areas within Minas Gerais, Brazil, and were evaluated at the out‐patient clinic of the Universidade Federal de Minas Gerais. These patients have been followed for more than 10 years, and repeated evaluations confirmed the clinical forms. In additional to standard serology examinations, physical examinations, electrocardiogram, chest X‐rays and echocardiogram were performed with the purpose of characterizing the clinical status of the patients, as defined previously 30. Indeterminate Chagas patients (IND; n = 6; age range = 34–69 years; four females and two males) had positive serology, lack of clinical manifestations or alterations upon clinical, radiological and echocardiographic examination. Cardiac Chagas patients (CARD; n = 6; age range = 18–72 years; four females and two males) displayed positive serology, right and/or left ventricular dilation, global left ventricular dysfunction, alterations in the cardiac electric impulse generation and conduction upon electrocardiogram, chest X‐rays and echocardiography. We excluded from our study individuals with any other chronic inflammatory diseases, diabetes, heart/circulatory illnesses or bacterial infections. All individuals included in this work were volunteers and treatment and clinical care was offered to all patients as needed, despite their enrollment into this research project. This cross‐sectional study is part of an extended project evaluating biomarkers of cardiomyopathy development in Chagas disease, which has the approval of the National Committee of Ethics in Research (CONEP#195/2007) and are in accordance with the Declaration for Helsinki.

Parasites and fractions

9. cruzitrypomastigotes of the CL Brener clone were grown in Vero cells, as performed previously by us 9. Briefly, cells were infected with 10 trypomastigotes/cell and, after removal of free trypomastigotes by washing with culture media, were maintained in RPMI enriched with 5% fetal calf serum and antibiotic (penicillin 500 U/ml and streptomycin 0·5 mg/ml) for approximately 6 days. After this period, trypomastigotes ruptured the cells and were collected from the supernatant, centrifuged, resuspended in phosphate‐buffered saline (PBS) and then washed twice with phosphate‐buffered saline (PBS) by centrifugation (800 gfor 5 min at 4°C). Parasites obtained in such a manner were stored at −80°C as dry pellets; 2 × 10⁹ tripomastigotes prepared as described previously were used for subsequent antigen fractionation.
10. cruzifractions were obtained using the methodology published previously 22, 31, with some changes. Briefly, frozen pellets obtained as described above were suspended in 1·6 ml of ultrapure water and transferred to 13 × 100‐mm polytetrafluoroethylene (PTFE)‐lined screw capped Pyrex culture tubes. Chloroform and methanol were added to each vial at a final ratio of chloroform/methanol/water (C/M/W) of 1 : 2 : 0.8 (v/v/v). Samples were mixed vigorously using a vortex for 2 min and then centrifuged for 15 min at 1800 gat room temperature; this process was repeated three times. Pellet was extracted once again with C/M (2 : 1, v/v), samples were mixed vigorously for 2 min using a vortex and then centrifuged for 15 min at 1800 g at room temperature. Insoluble tripomastigote debris were stored for later extraction of proteins. At the end of each step of extraction, the supernatants were transferred to PTFE‐lined Pyrex glass test tubes and then dried on a lyophilizer. Next, samples were pooled together and subjected to Folch's partition 32. Briefly, sample was dissolved in C/M/W (4 : 2 : 1·5, v/v/v) and then mixed vigorously for 5 min using a vortex and finally centrifuged for 15 min at 1800 g at room temperature. After centrifugation, a lower (organic) phase containing lipids and an upper (aqueous) phase containing glycolipids were obtained. Once again samples were dried by lyophilizing. The lipid fraction (LIP antigen, which contains mainly neutral membrane lipids) was resuspended in 1·0 ml of dimethylsulphoxide (DMSO). One millilitre of ultrapure water was added to the tube containing the glycolipids (GCL antigens). All fractions were in stored in sterile glass tubes at −80°C until use.

For T. cruzi protein extraction, we used the remaining insoluble material from lipid and glycolipid extraction, as described Almeida et al. (2000) 22. In brief, parasite debris was extracted three times with 10 volumes of butan–1–ol‐saturated water (9% butan–1–ol) for 4 h under agitation at room temperature. The resulting extracts were combined in a single vial and dried on the lyophilizer. Protein fraction extract (PRO antigen, which contains proteins and glycoproteins) was resuspended in 1·0 ml of ultrapure water and stored at −80°C until use.

Blood sampling and in‐vitro cultures

Peripheral blood samples were collected from all 18 volunteers enrolled into this study in heparin tubes. Peripheral blood mononuclear cells (PBMCs) were obtained by separating whole blood over Ficoll (Sigma Chemical Co., St Louis, MO, USA), as described previously by us 33. The cells were washed and resuspended in RPMI‐1640 medium (Gibco, Rockville, MD, USA) supplemented with 5% heat‐inactivated AB human serum (Sigma‐Aldrich, St Louis, MO, USA), antibiotics (penicillin, 200 U/ml and streptomycin, 0·1 mg/ml) and L‐glutamine (1 mM) (Sigma‐Aldrich) at a concentration of 1 × 10⁷ cells/ml. Cells from each volunteer were placed on 96‐well plates (Costar, Corning Incorporated, Corning, NY, USA) in 200 μl cultures under the following conditions: medium alone, GCL fraction (20 μg/ml), LIP fraction (equal to five parasites) and PRO fraction (equal to 10 parasites) for 14 and 36 h. The concentration of each stimulus in culture was determined based on previous tests performed by us using the expression of CD69 in DN T cells as a parameter of activation 34. GCL fraction was measured using the phenol–sulphuric acid method 35, while LIP and PRO fractions were extrapolated from the number of parasites used for the extraction. Brefeldin A (10 μg/ml) was added for the last 4 h of culture to prevent cytokine secretion.

Flow cytometric analysis

Immunophenotypical analyses of peripheral blood mononuclear cells (PBMCs) exposed to the different stimuli were performed by multi‐parametric flow cytometry to determine the activation status and cytokine production by the DN T cell subsets. Stimulated (treated as described above) or non‐stimulated cells were harvested after the final 18 or 40 h of culture and submitted to specific staining. We used combinations of monoclonal antibodies (mAbs) specific for human leucocyte cell‐surface markers, including: BV510‐labelled anti‐CD4, allophycocyanin‐cyanin 7 (APC‐Cy7)‐labelled anti‐CD8, BV421‐labelled anti‐TCR‐αβ and APC‐labelled anti‐TCR‐γδ to identify the specific subpopulations of DN T cells. A 40‐μl mixture of these surface antibodies was added to each well of a 96‐well round‐bottomed plate containing 2 × 10⁵ cells for 15 min at 4°C, washed in phosphate‐buffered saline (PBS) containing 1% bovine serum albumin (BSA), and fixed by a 20‐min incubation with a 2% formaldehyde solution. After removal of the fixing solution by centrifugation and washing with PBS, we permeabilized the cells by incubation for 15 min with a 0·5% saponin solution, and proceeded to intracellular staining. Samples were incubated with phycoerythrin (PE)‐Cy7‐labelled anti‐IL‐10 or CD69, PE‐labelled anti‐IFN‐γ or TNF monoclonal antibodies for 20 min at room temperature, washed twice with 0·5% saponin solution, resuspended in PBS and read in a flow cytometer. A minimum of 100 000 gated events from each sample were collected and analysed using FlowJo software (TreeStar, Inc., Ashland, OR, USA). All antibodies were from BioLegend (San Diego, CA, USA).

Statistical analysis

All data showed a Gaussian distribution, as determined by Kolmogorov–Smirnov test. Paired t‐test was used to ascertain differences between unstimulated cultures and stimulated cultures within the same group of patients. Comparisons between different groups were performed using unpaired t‐test. Differences that returned P‐values of less than or equal to 0·05 were considered statistically significant from one another.

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RESULTS

Glycolipid‐enriched fractions lead to increased activation of DN T cells from Chagas patients

We have shown previously that DN T cells from Chagas patients expand upon exposure to live T. cruzi trypomastigotes, and this expansion is accompanied by the production of immunoregulatory cytokines 16, 36. To determine which component(s) of T. cruzi trypomastigotes was (were) responsible for DN T cell activation, we fractionated the trypomastigote forms, as described above, and obtained GCL, LIP and PRO fractions. These fractions were used to stimulate PBMC from Chagas patients of the IND and CARD forms, as well as from non‐Chagas patients (healthy) as controls. As a measure of DN T cell activation, we evaluated the expression of the activation marker, CD69, by the TCR‐αβ and TCR‐γδ DN T cell subpopulations.

We observed that LIP and PRO fractions did not lead to a significant increase in the activation status of DN TCR‐αβ or DN TCR‐γδ cell subpopulations (Fig. ​(Fig.1a,b,1a,b, respectively) from Chagas patients or from non‐Chagas individuals, compared to non‐stimulated cultures. Conversely, GCL led to a significant increase in CD69 expression in DN TCR‐αβ cells from IND (Fig. ​(Fig.1a)1a) and by DN TCR‐γδ cells from IND, as well as CARD (Fig. ​(Fig.1b).1b). Expression of CD69 by DN T cells from non‐Chagas individuals did not return statistically significant results.

 

 

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Figure 1

Analysis of CD69 expression by double‐negative T cells (DN T) after exposure to different antigen fractions from CL Brener trypomastigotes. Cells from healthy individuals (n = 6), indeterminate (n = 6) and cardiac (n = 6) Chagas patients were incubated with media (MED), glycolipid (CGL), lipid (LIP) or protein (PRO)‐enriched fractions. The frequency of expression of CD69 was evaluated in the different DN T cell subpopulations, as described in Materials and methods. Panels (a) and (b) show the frequency of αβ and γδ DN T cells expressing CD69, respectively, under the different conditions. The bottom graphs of (a) and (b) are representative histograms from one donor from each of the groups, showing the intensity of expression of CD69 for the different conditions. Bars indicate averages and lines in each bar indicate standard deviation. Statistically significant results are indicated in the graphs.

Previous studies have shown that T. cruzi molecules can stimulate CD4⁺ and CD8⁺ T cells 37, 38, 39, 40. We then evaluated the expression of CD69 by CD4⁺ and CD8⁺ cells stimulated with GCL to determine whether we would also observe activation of these cells and, if so, if the magnitude of activation was similar to the one observed in DN T cells. We observed that GCL led to a slight increase in the activation of T cells from IND and CARD Chagas patients, especially CD4⁺ T cells (Fig. ​(Fig.2).2). CD4⁺ or CD8⁺ T cells from non‐Chagas individuals did not show up‐regulation of CD69 upon stimulation with GCL (Fig. ​(Fig.2).2). Interestingly, although the threshold of CD69 expression is already higher in DN T cells compared with CD4⁺ and CD8⁺ cells, as observed in non‐stimulated cultures, the magnitude of activation of DN T cells after GCL stimulation is at least three times higher than that observed in CD4⁺ and CD8⁺ T cells (Fig. ​(Fig.2).2). These data show that, although GCL can stimulate CD4⁺ and CD8⁺ cells, it stimulates DN T cells preferentially.

 

 

Figure 2

Expression of CD69 by CD4⁺, CD8⁺ and αβ⁺ double‐negative T cells (DN T) after stimulation with Trypanosoma cruzi‐derived glycoconjugate‐enriched fraction. Cells from healthy individuals (n = 6), indeterminate (n = 6) and cardiac (n = 6) Chagas patients were incubated with media (MED) or glycoconjugate (CGL)‐enriched fractions. The frequency of expression of CD69 by CD4⁺, CD8⁺ and CD4^–CD8^– cells was evaluated, as described in Materials and methods. Bars indicate averages and lines in each bar indicate standard deviation. Statistically significant results are indicated in the graphs.

Activation induced by glycolipid‐enriched fractions is accompanied by the expression of TNF and IFN‐γ by DN T cells from indeterminate and cardiac Chagas patients, respectively

To determine if the activation induced by the GCL was accompanied by a functional activation, we evaluated the expression of TNF, IFN‐γ and IL‐10 by the different DN T cell subpopulations. DN TCR‐αβ cells did not express statistically significant alterations in the frequency of cytokines upon stimulation with GCL in any of the groups (Fig. ​(Fig.3a–c),3a–c), despite the observed increase in CD69 expression in DN TCR‐αβ cells from IND (Fig. ​(Fig.1a).1a). The other stimuli (LIP and PRO) also did not induce significant changes in the expression of any of the evaluated cytokines by TCR‐αβ DN T cells from Chagas patients, regardless of the clinical form, or from non‐Chagas patients (data not shown). We observed that GCL induced a higher expression of TNF and IL‐10 (Fig. ​(Fig.3d,f)3d,f) in DN TCR‐γδ cells from IND, while inducing an increase in the expression of IFN‐γ in DN T cells from CARD (Fig. ​(Fig.33e).

 

 

Figure 3

Analysis of tumour necrosis factor (TNF), interferon (IFN)‐γ and interleukin (IL)‐10 expression by double‐negative T cells (DN T) after exposure to glycoconjugate (CGL) fraction from CL Brener trypomastigotes. Cells from healthy individuals (n = 6), indeterminate (n = 6) and cardiac (n = 6) Chagas patients were incubated with media (MED) or CGL‐enriched fractions. The frequency of expression of TNF, IFN‐γ and IL‐10 was evaluated in the different DN T cell subpopulations, as described in Materials and methods. (a,b,c) The frequency of TNF, IFN‐γ and IL‐10 in αβ DN T cells; (d,e,f) The frequency of TNF, IFN‐γ and IL‐10 in γδ DN T cells, under the different conditions. Bars indicate averages and lines in each bar indicate standard deviation. Statistically significant results are indicated in the graphs.

To determine if CGL stimulation led to changes in the balance between the expression of inflammatory and anti‐inflammatory cytokines in the DN TCR‐γδ cells, which were stimulated preferentially by GCL, we calculated the ratio between the percentage of DN TCR‐γδ cells expressing TNF/IL‐10 and IFN‐γ/IL‐10 before and after stimulation with GCL. Our analysis showed that the TNF/IL‐10 ratio in DN TCR‐γδ cells did not change significantly after GCL stimulation in any of the analysed groups compared to non‐stimulated cultures (Fig. ​(Fig.4).4). Conversely, while we observed a slight decrease in the IFN‐γ/IL‐10 ratio in DN TCR‐γδ cells from IND after stimulation with GCL compared to media control, this ratio was increased significantly in DN TCR‐γδ cells from CARD (Fig. ​(Fig.4).4). Moreover, the IFN‐γ/IL‐10 ratio in DN TCR‐γδ cells from CARD after GCL stimulation was statistically greater than the one from non‐Chagas individuals stimulated with GCL (Fig. ​(Fig.44).

 

 

 

Figure 4

Tumour necrosis factor (TNF)/interleukin (IL)‐10 and interferon (IFN)‐γ/IL‐10 ratio in T cell receptor (TCR) αβ⁺ double‐negative T cells (DN T) from non‐infected, indeterminate and cardiac patients before and after glycolipid (GCL) stimulation. Cells from healthy individuals (n = 6), indeterminate (n = 6) and cardiac (n = 6) Chagas patients were incubated with media (MED) or CGL‐enriched fractions. The ratios of % TNF⁺/IL‐10⁺ and IFN‐γ⁺/IL‐10⁺ DN γδ⁺ T cells are expressed in the left and right y‐axis, respectively. Bars indicate averages and lines in each bar indicate standard deviation. Statistically significant results are indicated in the graphs.

Glycolipid‐enriched fraction induces cytokine expression by CD8⁺ T lymphocytes from indeterminate patients

CD8⁺ T lymphocytes are the predominant cells present in the inflammatory infiltrate in damaged heart tissue from cardiac Chagas patients. Our results showed that the GCL fraction induced an increase in TNF and IL‐10 expression in CD8⁺ T cells from IND (Fig. ​(Fig.5a,c).5a,c). GCL stimulus did not affect the production of cytokines by CD8⁺ cells from CARD or healthy individuals (Fig. ​(Fig.5a–c).5a–c). Expression of IFN‐γ by CD8⁺ cells was not altered after GCL stimulation in any of the analysed groups (Fig. ​(Fig.5b).5b). The glycolipid‐enriched fraction did not alter the functional activity of CD4⁺ T lymphocytes from healthy, IND or CARD (Fig. 5d–f).

 

Figure 5

Analysis of tumour necrosis factor (TNF), interferon (IFN)‐γ and interleukin (IL)‐10 expression by CD4⁺ and CD8⁺ T cells after exposure to glycolipid (GCL) fractions from CL Brener trypomastigotes. Cells from healthy individuals (n = 6), indeterminate (n = 6) and cardiac (n = 6) Chagas patients were incubated with media (MED) or CGL‐enriched fractions. The frequency of expression of TNF, IFN‐γ and IL‐10 was evaluated in CD8⁺ and CD4⁺ T cell subpopulations, as described in Materials and methods. (a,b,c) The frequency of TNF, IFN‐γ and IL‐10 in CD8⁺ T cells; (d,e,f) The frequency of TNF, IFN‐γ and IL‐10 in CD4⁺ T cells, under the different conditions. Bars indicate averages and lines in each bar indicate standard deviation. Statistically significant results are indicated in the graphs.

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DISCUSSION

The T. cruzi surface is decorated with a large variety of molecules that, in addition to participating in the host–parasite interaction, display antigenic characteristics. Among the main components of the T. cruzi cell surface, glycoconjugates have been studied widely, especially GIPLs and GPI mucins. It has been shown that they mediate cellular invasion and evasion of the immune system directly 41. Studies in the experimental model of T. cruzi infection also showed that these molecules display a proinflammatory activity, inducing the expression of inflammatory cytokines by macrophages and T cells 22, 42. GIPLs, a class of GCL, from various parasites such as T. cruzi, Toxoplasma gondii and Plasmodium falciparum can induce the production of proinflammatory cytokines mediated by TLR‐4 26, 43, 44. However, by reducing the expression of co‐stimulatory and activation molecules, as well as cytokine production, a modulatory effect of GIPLs on human macrophages and dendritic cells has also been demonstrated 45.

In recent years, DN T cells have emerged as important sources of immunoregulatory cytokines in infectious diseases such as leishmaniasis, tuberculosis and Chagas disease 16, 36, 46, 47. In Chagas disease, these cells express inflammatory and anti‐inflammatory cytokine profiles, associated with the cardiac and indeterminate forms, respectively 16. The mechanisms of activation of these cells, as well as the molecules responsible for their activation, have not yet been elucidated. In this work, we studied the ability of different components of T. cruzi in stimulating DN T cells. We observed that the glycolipid‐enriched fraction (GCL) is the most important component from T. cruzi in the activation of DN T cells, while lipid (LIP)‐ and protein (PRO)‐ enriched fractions are not efficient stimulators of DN T cells from Chagas patients. Moreover, we observed that GCL induces a higher expression of TNF and IL‐10 in cells from indeterminate patients (IND), while the expression of IFN‐γ was increased only in cells from cardiac Chagas patients (CARD). Importantly, the TNF/IL‐10 ratio did not change significantly indicating that, although TNF was induced by GCL in DN T cells from IND, it was not enough to induce a more inflammatory profile in these patients. Conversely, the IFN‐γ/IL‐10 ratio increased in CARD, indicating that the magnitude of the increase in IFN‐γ expression induced by GCL stimulation influenced the cytokine milieu. Although GCL also stimulates CD4⁺ and CD8⁺ T cells, the magnitude of this stimulation is significantly greater in DN T cells. Thus, our data show that GCL is a potent activator of DN T cells, especially TCR‐γδ, and that this activation is responsible for the induction of an inflammatory environment in DN T cells from CARD, but not IND. Thus, modulating the inflammatory response of DN T cells to GCL may emerge as an alternative approach for avoiding or, at least, controlling Chagas cardiac pathology.

One of the measures of activation used in our work was the expression of CD69. We observed that GCL‐stimulated DN T cells from CARD and IND, but not from non‐Chagas patients, expressed CD69. This activation was observed mainly in DN TCR‐γδ cells compared to the αβ subpopulation and, although CD4⁺ and CD8⁺ T cells also were slightly activated, the magnitude of their activation was much lower than that of DN T cells. Medeiros et al. (2007) 48 observed that, as a result of activation of mouse macrophages treated with GIPLs, T and B lymphocytes from the spleen and lymph nodes of these animals displayed increased CD69 expression, showing an indirect effect of GIPL stimulation on these cells. In our study, the fact that the activation was observed only in DN TCR‐γδ cells and only slightly in CD4⁺ and CD8⁺ T cells, suggests that this was a direct stimulation of DN TCR‐γδ cells by GCL.

The reasons why most of the response of DN T cells to GCL is concentrated in the TCR‐γδ subpopulation are not clear. However, several studies have shown that αβ and γδ DN subpopulations display a distinct repertoire and activation requirements. While DN TCR‐αβ cells display a polyclonal repertoire with MHC restriction 17, 49, 50, DN TCR‐γδ cells display a somewhat oligoclonal repertoire 18 which could explain why certain preparations, such as GCL, stimulate one population more effectively than the other. It is possible that this stimulation occurs via activation of Toll‐like receptor agonists present in the GCL preparation. This is an important point that merits future investigation. That GCL activates DN TCR‐γδ cells preferentially may prove beneficial for implementation of immunomodulatory treatments that block the response of DN TCR‐γδ cells to GCL in attempts to control inflammation and, thus, pathology development in Chagas disease. The ability to interfere with one population individually and not affect the response of others may offer an additional advantage toward the use of this strategy.

Previous studies have shown that DN T cells respond to lipid antigens presented via CD1 molecules 51, 52. It is puzzling that the T. cruzi‐derived LIP fraction did not induce activation of DN T cells in our study. However, it is important to note that most lipids present in the LIP‐enriched fraction are neutral lipids 31 and possibly with less immunogenic potential. In addition, GCL fraction is indeed the fraction that contains highly immunogenic lipids such as GIPLs and possibly others, which explains its stimulatory capacity over DN T cells. Previous studies have also shown that GIPLs can lead to inflammatory, but also anti‐inflammatory, responses in human cells 22, 42, 45. Here, we observed that GCL led to inflammatory and anti‐inflammatory responses in DN T cells from CARD and IND, respectively. Given the complexity of the GCL fraction, it is possible that DN T cells from IND and CARD are activated by different components, leading to mobilization of distinct stimulatory pathways. Alternatively, if the cells from CARD and IND are responding to a dominant antigen, the difference in the response could be a result of the overall inflammatory and anti‐inflammatory milieu observed in these patients, respectively, possibly associated to a different priming/response in patients with different clinical forms. Previous studies have shown that GCL from trypomastigote forms of the CL Brener clone, which was used in this study, include mainly alkylacylglycerol and dihydroceramide‐containing GIPL 42, but that it also may contain other components such as phosphoinositols and inositol phospatidylcholine 53. Given the high immunogenicity of the different GIPLs, molecular characterization of the GCL fraction, as well as testing of its individual constituents, need to be performed to identify precisely the component(s) responsible for this differential activation in IND and CARD. This analysis will be instrumental to aid in developing tools to control the response to GCL as a possible strategy to block or control pathology.

Our study employs components derived from the trypomastigote form of T. cruzi, which provides important information towards defining the activating ability of molecules derived from a parasite stage that is found in the host. Because of its role in activating the immune response, GPI mucins and GIPLs are being used as adjuvants in immunization protocols against T. cruzi, with the hope of finding effective vaccines 54. To date, the efforts have not led to highly effective protective responses. Our study emphasizes the importance of considering the response mounted by DN T cells in vaccination efforts, given their prominent response to GIPL, which may influence the success (or not) of a given vaccine.

In conclusion, our study shows that GCL are the main fraction of T. cruzi responsible for the activation of DN TCR‐γδ cells in Chagas patients, and for the induction of an inflammatory profile in cardiac but not indeterminate patients. Thus, considering the possible influence of this activation in the overall response of the patients, which is yet to be evaluated, it is possible that the control of DN TCR‐γδ cells in Chagas patients may emerge as a potentially preventive inducer of pathology development. Determining the fine molecular composition of GCL, as well as the mechanisms of GCL‐induced activation, will clarify important points towards defining possible strategies to modulate the inflammatory response in cardiac Chagas patients.

Funding: Fundação de Amparo a Pesquisa do Estado de Minas Gerais, Universal 2014; Conselho Nacional de Desenvolvimento Científico e Tecnologico, INCT‐DT 2016.

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DISCLOSURE

The authors have no conflicts of interest.

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ACKNOWLEDGEMENTS

1. S. A. P. conducted the experiments, analysis and data interpretation; L. M. D. M. collaborated in the execution of the experiments and parasite culture; R. P. S. and A. F. M. contributed to antigen fractionation efforts; M. C. P. N. was the clinician responsible for the care, classification and enrolment of the patients; K. J. G. and W. O. D. contributed to experimental design, analysis and data interpretation.

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REFERENCES

1. Dutra WO, Menezes CAS, Magalhães LMD, Gollob KJ. Immunoregulatory networks in human Chagas disease. Parasite Immunol 2014; 36:377–87. [PMC free article] [PubMed] [Google Scholar]
2. Nunes MC, Dones W, Morillo C, Encina J, Ribeiro L. Chagas disease: an overview of clinical and epidemiological aspects. J Am Coll Cardiol 2013; 62:767–76. [PubMed] [Google Scholar]
3. Lee BY, Bacon KM, Bottazzi ME, Hotez PJ. Global economic burden of Chagas disease: a computational simulation model. Lancet Infect Dis 2013; 13:342–8. [PMC free article] [PubMed] [Google Scholar]
4. Dutra WO, Gollob KJ. Current concepts in immunoregulation and pathology of human Chagas disease. Curr Opin Infect Dis 2008; 21:287–92. [PMC free article] [PubMed] [Google Scholar]
5. Cunha‐Neto E, Chevillard C. Chagas disease cardiomyopathy: immunopathology and genetics. Mediat Inflamm 2014; 2014:683230. [PMC free article] [PubMed] [Google Scholar]
6. Talvani A, Rocha MO, Barcelos LS, Gomes YM, Ribeiro AL, Teixeira MM. Elevated concentrations of CCL2 and tumor necrosis factor‐alpha in chagasic cardiomyopathy. Clin Infect Dis 2004; 38:943–50. [PubMed] [Google Scholar]
7. Gomes JAS, Bahia‐Oliveira LMG, Rocha MOC, Martins‐Filho OA, Gazzinelli G, Correa‐Oliveira R. Evidence that development of severe cardiomyopathy in human Chagas' disease is due to a Th1‐specific immune response. Infect Immun 2003; 71:1185–93. [PMC free article] [PubMed] [Google Scholar]
8. Cunha‐Neto E, Dzau VJ, Allen PD et alCardiac gene expression profiling provides evidence for cytokinopathy as a molecular mechanism in Chagas' disease cardiomyopathy. Am J Pathol 2005; 167:305–13. [PMC free article] [PubMed] [Google Scholar]
9. Souza PE, Rocha MOC, Rocha‐Vieira E et alMonocytes from patients with indeterminate and cardiac forms of Chagas' disease display distinct phenotypic and functional characteristics associated with morbidity. Infect Immun 2004; 72:5283–91. [PMC free article] [PubMed] [Google Scholar]
10. Costa GC, da Costa Rocha MO, Moreira PR, Menezes CA, Silva MR, Gollob KJ, Dutra WO. Functional IL‐10 gene polymorphism is associated with Chagas disease cardiomyopathy. J Infect Dis 2009;199(3):451–4. [PubMed] [Google Scholar]
11. Magalhães LMD, Villani FNA, Nunes MCP, Gollob KJ, Rocha MOC, Dutra WO. High interleukin 17 expression is correlated with better cardiac function in human Chagas disease. J Infect Dis 2013; 207:661–5. [PMC free article] [PubMed] [Google Scholar]
12. Guedes PM, Gutierrez FR, Silva GK et alDeficient regulatory T cell activity and low frequency of IL‐17‐producing T cells correlate with the extent of cardiomyopathy in human Chagas' disease. PLOS Negl Trop Dis 2013; 6:e1630. [PMC free article] [PubMed] [Google Scholar]
13. Souza PE, Rocha MO, Menezes CA et alTrypanosoma cruzi infection induces differential modulation of costimulatory molecules and cytokines by monocytes and T cells from patients with indeterminate and cardiac Chagas' disease. Infect Immun 2007; 75:1886–94. [PMC free article] [PubMed] [Google Scholar]
14. Menezes CA, Rocha MO, Souza PE, Chaves AC, Gollob KJ, Dutra WO. Phenotypical and functional characteristics of CD28⁺and CD28^– cells from chagasic patients: distinct repertoire and cytokine expression. Clin Exp Immunol 2004; 137:129–38. [PMC free article] [PubMed] [Google Scholar]
15. de Araújo FF, Corrêa‐Oliveira R, Rocha MO et alFoxp3+CD25 (high) CD4+regulatory T‐cells from indeterminate patients with Chagas disease can suppress the effector cells and cytokines and reveal altered correlations with disease severity. Immunobiology 2012; 217:768–77. [PubMed] [Google Scholar]
16. Villani FN, Costa‐Rocha MO, Nunes MD et alTrypanosoma cruzi induced activation of functionally distinct αβ and γδ CD4–CD8– T cells in individuals with polar forms of Chagas' disease. Infect Immun 2010; 78:4421–30. [PMC free article] [PubMed] [Google Scholar]
17. Fischer K, Voelkl S, Heymann J et alIsolation and characterization of human antigen specific TCR alpha beta+ CD4−CD8− double‐negative regulatory T cells. Blood 2004; 105:2828–35. [PubMed] [Google Scholar]
18. Carding SR, Egan PJ. Gammadelta T cells: functional plasticity and heterogeneity. Nat Rev Immunol 2002; 2:336–45. [PubMed] [Google Scholar]
19. Nogueira NP, Saraiva FMS, Sultano PE et alProliferation and differentiation of Trypanosoma cruzi inside its vector have a new trigger: redox status. PLOS ONE 2015; 10:e0116712. [PMC free article] [PubMed] [Google Scholar]
20. Bayer‐Santos E, Aguilar‐Bonavides C, Rodrigues SP et alProteomic analysis of Trypanosoma cruzi secretome: characterization of two populations of extracellular vesicles and soluble proteins. J Proteome Res 2013; 12:883–97. [PubMed] [Google Scholar]
21. Camargo MM, Almeida IC, Pereira ME, Ferguson MAJ, Travassos LR, Gazzinelli RT. Glycosylphosphatidylinositol‐anchored mucin‐like glycoproteins isolated from Trypanosoma cruzitrypomastigotes initiate the synthesis of proinflammatory cytokines by macrophages. J Immunol 1997; 158:5890–901. [PubMed] [Google Scholar]
22. Almeida IC, Camargo MM, Procopio DO et alHighly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. EMBO J 2000; 19:1476–85. [PMC free article] [PubMed] [Google Scholar]
23. Bartholomeu DC, Cerqueira GC, Leão AC et alGenomic organization and expression profile of the mucin‐associated surface protein (masp) family of the human pathogen Trypanosoma cruzi . Nucleic Acids Res 2009; 37:3407–17. [PMC free article] [PubMed] [Google Scholar]
24. Soares RP, Torrecilhas AC, Assis RR et alIntraspecies variation in Trypanosoma cruzi GPI‐mucins: biological activities and differential expression of alpha‐galactosyl residues. Am J Trop Med Hyg 2012; 87:87–96. [PMC free article] [PubMed] [Google Scholar]
25. Campos MA, Almeida IC, Takeuchi O et alActivation of Toll‐like receptor‐2 by glycosylphosphatidylinositol anchors from a protozoan parasite. J Immunol 2001; 167:416–23. [PubMed] [Google Scholar]
26. Oliveira AC, Peixoto JR, de Arruda LB et alExpression of functional TLR4 confers proinflammatory responsiveness to Trypanosoma cruzi glycoinositolphospholipids and higher resistance to infection with T. cruzi . J Immunol 2004; 173:5688–96. [PubMed] [Google Scholar]
27. Gazzinelli RT, Denkers EY. Protozoan encounters with Toll‐like receptor signalling pathways: implications for host parasitism. Nat Rev Immunol 2006; 6:895–906. [PubMed] [Google Scholar]
28. Dutra WO, Menezes CA, Villani FN et alCellular and genetic mechanisms involved in the generation of protective and pathogenic immune responses in human Chagas disease. Mem Inst Oswaldo Cruz 2009; 1:208–18. [PMC free article] [PubMed] [Google Scholar]
29. Machado FS, Dutra WO, Esper L et alCurrent understanding of immunity to Trypanosoma cruzi infection and pathogenesis of Chagas disease. Semin Immunopathol 2012; 34:753–70. [PMC free article] [PubMed] [Google Scholar]
30. Ribeiro SM, Morceli J, Gonçalves RS et alAccuracy of chest radiography plus electrocardiogram in diagnosis of hypertrophy in hypertension. Arq Bras Cardiol 2012; 99:825–33. [PubMed] [Google Scholar]
31. Gazos‐Lopes F, Oliveira MM, Hoelz LV et alStructural and functional analysis of a platelet‐activating lysophosphatidylcholine of Trypanosoma cruzi . PLOS Negl Trop Dis 2014; 8:e3077. [PMC free article] [PubMed] [Google Scholar]
32. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957; 226:4. [PubMed] [Google Scholar]
33. Bottrel RL, Dutra WO, Martins FA et alFlow cytometric determination of cellular sources and frequencies of key cytokine producing lymphocytes directed against recombinant LACK and soluble Leishmania antigen in human cutaneous leishmaniasis. Infect Immun 2001; 69:3232–9. [PMC free article] [PubMed] [Google Scholar]
34. Ziegler SF, Ramsdell F, Alderson MR. The activation antigen CD69. Stem Cells 1994; 12:456–65. [PubMed] [Google Scholar]
35. Rao P, Pattabiraman TN. Reevaluation of the phenol–sulfuric acid reaction for the estimation of hexoses and pentoses. Anal Biochem 1989; 181:18–22. [PubMed] [Google Scholar]
36. Passos LS, Villani FN, Magalhães LM et alBlocking of CD1d decreases Trypanosoma cruzi‐induced activation of CD4–CD8– T cells and modulates the inflammatory response in patients with Chagas heart disease. J Infect Dis 2016; 266:1–10. [PubMed] [Google Scholar]
37. Gomes NA, Previato JO, Zingales B, Mendonça‐Previato L, DosReis GA. Down‐regulation of T lymphocyte activation in vitroand in vivo induced by glycoinositolphospholipids from Trypanosoma cruzi . J Immunol 1996; 156:628–35. [PubMed] [Google Scholar]
38. Bellio M, Liveira AC, Mermelstein CS et alCostimulatory action of glycoinositolphospholipids from Trypanosoma cruzi: increased interleukin 2 secretion and induction of nuclear translocation of the nuclear factor of activated T cells 1. FASEB J 1999; 13:1627–36. [PubMed] [Google Scholar]
39. Alvarez MG, Postan M, Weatherly DB et alHLA Class I‐T Cell epitopes from trans‐sialidase proteins reveal functionally distinct subsets of CD8+ T Cells in chronic Chagas disease. PLOS Negl Trop Dis 2008; 2:e288. [PMC free article] [PubMed] [Google Scholar]
40. Freire‐de‐Lima L, Alisson‐Silva F, Carvalho ST et alTrypanosoma cruzi subverts host cell sialylation and may compromise antigen‐specific CD8+ T cell responses. J Biol Chem 2010; 285:13388–96. [PMC free article] [PubMed] [Google Scholar]
41. Yoshida N. Molecular basis of mammalian cell invasion by Trypanosoma cruzi. An Acad Bras Cienc 2006; 78:87–111. [PubMed] [Google Scholar]
42. Previato JO, Wait R, Jones C et alGlycoinositolphospholipid from Trypanosoma cruzi: structure, biosynthesis and immunobiology. Adv Parasitol 2004; 56:1–41. [PubMed] [Google Scholar]
43. Debierre‐Grockiego F, Azzouz N, Schmidt J et alRoles of glycosylphosphatidylinositols of Toxoplasma gondii. Induction of tumor necrosis factor‐alpha production in macrophages. J Biol Chem 2003; 278:32987–93. [PubMed] [Google Scholar]
44. Krishnegowda G, Hajjar AM, Zhu J et alInduction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem 2005; 280:8606–16. [PMC free article] [PubMed] [Google Scholar]
45. Brodskyn C, Patricio J, Oliveira R et alGlycoinositolphospholipids from Trypanosoma cruzi interfere with macrophages and dendritic cell responses. Infect Immun 2002; 70:3736–43. [PMC free article] [PubMed] [Google Scholar]
46. Antonelli LR, Dutra WO, Almeida RP, Bacellar O, Carvalho EM, Gollob KJ. Activated inflammatory T cells correlate with lesion size in human cutaneous leishmaniasis. Immunol Lett 2005; 101:226–30. [PubMed] [Google Scholar]
47. Pinheiro MB, Antonelli LR, Sathler‐Avelar R et alCD4‐CD8‐αβ and γδ T cells display inflammatory and regulatory potentials during human tuberculosis. PLOS ONE 2012; 7:e50923. [PMC free article] [PubMed] [Google Scholar]
48. Medeiros MM, Peixoto JR, Oliveira AC et alToll‐like receptor 4 (TLR4)‐dependent proinflammatory and immunomodulatory properties of the glycoinositolphospholipid (GIPL) from Trypanosoma cruzi . J Leukoc Biol 2007; 82:488–96. [PubMed] [Google Scholar]
49. Leprince AB, Mateo V, Lim A et alHuman TCR alpha/beta+ CD4–CD8– double‐negative T cells in patients with autoimmune lymphoproliferative syndrome express restricted Vbeta TCR diversity and are clonally related to CD8+ T cells. J Immunol 2008; 181:440e8. [PubMed] [Google Scholar]
50. Ford MS, Chen W, Wong S et alPeptide‐activated double‐negative T cells can prevent autoimmune type‐1 diabetes development. Eur J Immunol 2007; 37:2234e41. [PubMed] [Google Scholar]
51. Amprey J, Im J, Turco S et alliver NK T cells is activated during Leishmania donovani infection by CD1d‐bound lipophosphoglycan. J Exp Med 2004; 200:895–904. [PMC free article] [PubMed] [Google Scholar]
52. Donovan MJ, Jayakumar A, McDowell MA. Inhibition of groups 1 and 2 CD1 molecules on human dendritic cells by Leishmania species. Parasite Immunol 2007; 29:515–24. [PubMed] [Google Scholar]
53. Nakayasu ES, Yashunsky DV, Nohara LL, Torrecilhas AC, Nikolaev AV, Almeida IC. GPIomics: global analysis of glycosylphosphatidylinositol‐anchored molecules of Trypanosoma cruzi. Mol Syst Biol 2009; 5:261. [PMC free article] [PubMed] [Google Scholar]
54. Junqueira C, Guerrero AT, Galvão‐Filho B et alTrypanosoma cruzi adjuvants potentiate T cell‐mediated immunity induced by a NY‐ESO‐1 based antitumor vaccine. PLOS ONE 2012; 7:e36245. [PMC free article] [PubMed] [Google Scholar]

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