Regulatory T-cell frequency and function in acute myocardial infarction patients and its correlation with ventricular dysfunction

Abstract

A high percentage of patients with acute coronary syndrome develop heart failure due to the ischemic event. Regulatory T (Treg) cells are lymphocytes with suppressive capacity that control the immune response and include the conventional CD4+ CD25hi Foxp3+ cells and the CD4+ CD25var CD69+ LAP+ Foxp3− IL-10+ cells. No human follow-up studies focus on Treg cells’ behavior after infarction and their possible relationship with ventricular function as a sign of postischemic cardiac remodeling. This study aimed to analyze, by flow cytometry, the circulating levels of CD69+ Treg cells and CD4+ CD25hi Foxp3+ cells, their IL-10+ production as well as their function in patients with acute myocardial infarction (AMI), and its possible relation with ventricular dysfunction. We found a significant difference in the percentage of CD4+ CD25hi Foxp3+ cells and IL-10+ MFI in patients with AMI at 72 hours compared with the healthy control group, and the levels of these cells were reduced 6 months post-AMI. Regarding the suppressive function of CD4+ CD25+ regulatory cells, they were dysfunctional at 3 and 6 months post-AMI. The frequency of CD69+ Treg cells was similar between patients with AMI at 72 hours postinfarction and the control groups. Moreover, the frequency of CD69+ Treg cells at 3 and 6 months postischemic event did not vary over time. Treg cells play a role in regulating inflammation after an AMI, and its function may be compromised in this pathology. This work is the first report to evaluate CD69+ Foxp3− Treg cells in AMI patients.

A high percentage of patients with acute coronary syndrome develop heart failure due to the ischemic event. Regulatory T cells play a role in regulating inflammation after an acute myocardial infarction and its function may be compromised in this pathology. This work is the first report to evaluate CD69+ Foxp3− Treg cells in acute myocardial infarction patients.

Graphical Abstract

Graphical Abstract.

Introduction

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide, approximately 17.9 million people died from CVDs in 2019, and 85% of these deaths are due to heart attack and stroke [1].

Despite the improvement in diagnosis and treatment of patients with acute coronary syndrome (ACS), a high percentage of them develop heart failure due to the ischemic event. Acute myocardial infarction (AMI) occurs when an atherosclerotic plaque rupture or erosion occurs, inducing thrombus formation, which completely occludes the artery lumen cutting off blood supply to the myocardium. This ischemic event leads to an inflammatory process in the infarcted heart characterized by an infiltration of lymphocytes, monocytes, macrophages, and neutrophils, as well as by the production of high levels of TNFα, IL-1β, and members of the IL-6 family [2].

This inflammatory phase, which occurs in the first 72 hours, includes phagocytosis of necrotic cells and debris and constitutes an early physiological remodeling phase required for wound healing. Then, a resolution phase, late physiological remodeling, occurs, where predominant cells are those with anti-inflammatory properties, such as reparative monocytes, macrophages M2, and regulatory T (Treg) cells recruited by C–C chemokine receptor type 5 [3]. This resolution phase can last for several months and is necessary to form a stable scar composed of collagen, aiming to stabilize the damaged area and prevent future deformations. If the switch between both phases does not occur normally, the persistent and exacerbated inflammation leads to adverse ventricular remodeling. This pathological remodeling consists of changes in ventricular size, shape, and function. These changes may compromise ventricular contraction leading to a reduced ejection fraction and heart failure [4].

As mentioned above, the formation of a collagenous scar is necessary to heal the injured myocardium. This new collagenous matrix is produced by myofibroblasts and by the action of transforming growth factor-β (TGF-β). This cytokine contributes to tissue regeneration due to its ability to promote fibroblast differentiation into myofibroblasts, increasing interstitial fibrillar collagen synthesis in these cells. It has been observed in mice treated with CD28-superagonist that both activated Treg cells and M2 macrophages produce increased levels of TGF-β in the healing myocardium after induced an infarction by ligation of a coronary artery, compared with control sham animals [5]. Besides, in heart-draining lymph nodes from mice, along the first week after induction of infarction by ligation of a coronary artery, T cells CD4⁺ and Treg cells CD4⁺ Foxp3⁺ are activated by the antigen recognition by their TCR specific for cardiac auto-antigens, which facilitates the healing of the myocardium injury [6].

Treg cells are lymphocytes with suppressive capacity that inhibit immune responses; they are crucial for maintaining immunological homeostasis and self-tolerance, preventing autoimmunity [7]. The average levels in human peripheral blood are between 5% and 10% of CD4⁺ T cells [8]. Treg cells can be broadly classified into natural Treg cells (nTreg) or peripheral Treg cells (pTreg). nTreg develop and emigrate from the thymus, while pTreg are generated in the periphery from naive CD4⁺ lymphocytes. Both populations are characterized by the expression of CD25 (interleukin-2 receptor α chain) and the transcription factor, Foxp3, which is necessary for their development and function [9].

Regulatory CD4⁺ CD25⁺ T cells exert their suppressive function through different mechanisms, including inhibitory molecules such as CTLA-4 (CD152), which is constitutively expressed in Treg cells and interacts with the co-receptors expressed in antigen-presenting cells (APC) and activated T cells, CD80 and CD86. Cell contact-independent mechanisms involve inhibitory cytokine production such as IL-10 and TGF-β that have potent immunosuppressive properties, mediating suppression, or generating a suppressive milieu; for example, inducing the generation of pTreg [7]. TGF-β can exert its inhibitory effects in a soluble or membrane-bound manner [10], the last one associated with the latent associated peptide (LAP) [11]. IL-10 is a cytokine that can inhibit the pro-inflammatory cytokine synthesis by T cells and innate immunity cells. IL-10 has a crucial role in regulating inflammatory processes produced by pathogens or foreign particles and contributes to the prevention of atherosclerosis and decreasing plaque vulnerability [12].

Peripheral Treg cells may or may not express the transcription factor Foxp3. IL-10-secreting T regulatory 1 cells (Tr1) and TGF-β-secreting T helper cells (Th3) are examples of the second group. Furthermore, new populations have been recently described according to the expression of specific cell surface molecules. In this regard, a different subpopulation of Treg cells with a characteristic phenotype has been reported; these cells are CD4⁺ CD25^variable CD69⁺ LAP⁺ Foxp3^- IL-10⁺. CD69 is the earliest activation cell surface marker on leukocytes, transiently expressed on lymphocytes after activation but not in their basal state. CD69 participates in the maintenance of suppressive function and immune tolerance by Foxp3⁺ Treg cells maintaining high expression of suppression-associated markers [13], such as membrane-bound TGF-β by activating ERK, being suggested as an immunoregulatory receptor with effects on TGF-β synthesis [14].

In healthy subjects, the percentages of these cells in the peripheral blood range from near to zero values to 1.5% approximately [15] and in scenarios such as overweight in humans, its suppressive function is diminished [16].

Despite there are significant advances in understanding the role of Treg cells in different CVD, their participation in the biphasic immune response post-AMI, inflammatory and resolutive, remains unclear. Weirather et al. [5] observed increased CD4⁺ Foxp3⁺ Treg cells in cardiac tissue and mediastinal lymph nodes in mice post-MI compared with sham-operated mice. They also found that the absence of Treg cells resulted in aggravated cardiac inflammation because these cells modulate macrophage differentiation into an M2 reparative phenotype.

Bansal et al. [17] found in AMI murine models increased Treg cell levels in the heart and circulation after the induced infarction, and after the wound healing early phase, this could be possible because the myocardial milieu favors local differentiation of antigen-specific Treg lymphocytes [18]. In addition, these Treg cells expressed IFN-γ and TNF-α, and loss their immunosuppressive capacity. Furthermore, in patients with ACS, CD4⁺ CD25⁺ Treg cell levels are reduced, and their function is compromised, compared with patients with stable angina and normal artery subjects [19]. Within the first 3 days after AMI, the Treg cell density increases exponentially in infarcted myocardium and subsequently decreases. Tang et al. [20] observed that in heart failure patients, circulating Treg cell frequencies were lower than control subjects; these cells were also less effective in inhibiting cytokine synthesis. Even more, a positive correlation between Treg levels and LVEF (left ventricular ejection fraction) was observed; however, they do not evaluate the evolution time of heart failure.

Despite several studies in mice showing that Treg cells become activated after AMI and have a fundamental role in resolving ischemic injury, there are not enough studies in humans about this population’s role in the development of postischemic cardiac remodeling. Because of their potent immunosuppressive capacity contributing to inflammation resolution, Treg cells appear critical in cardiac healing. Studies in humans show lower numbers of these cells in patients with AMI. However, it is relevant to study the correlation between circulating Treg cell levels, conventional and CD69⁺, and their function in patients with AMI and correlate these parameters with ventricular dysfunction as indicative of heart failure.

Materials and methods

Patients

We included 19 patients with ST-segment elevation myocardial infarction. All patients were diagnosed according to clinical and laboratory criteria. Patients were recruited from the cardiology department of Hospital Central ‘Ignacio Morones Prieto’. The first sample was obtained immediately or within 72 hours after AMI from 19 patients. A second and a third sample were obtained from some of these patients, 3 (n = 12) and 6 months (n = 8) after the event. The echocardiographic valuation was performed at 72 hours, 3, and 6 months post-AMI. A control group included patients with cardiovascular risk (CVR; type 2 diabetes mellitus, arterial hypertension, dyslipidemia, and smoking) without AMI to compare the behavior of Treg lymphocytes after the acute ischemic event between groups with similar risk factors. A second control group was included and consisted of healthy subjects. Clinical and demographic data are summarized in Table 1. After explaining the project to the subjects, informed consents were obtained in all cases. The local University Ethics Committee (UASLP) approved this study (approval number 58-14). This work was carried out following The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.

Table 1.

Demographic and clinical data of AMI patients and control subjects

	AMI	CVR control	Healthy control
n	19	6	8
Age (mean ± SD)	63 ± 11.85	65.5 ± 9.02	44.75 ± 9.114
Sex	M: 16, F: 3	M: 5, F: 1	M: 4, F: 4
Type 2 diabetes mellitus	6	2	0
Hypertension	9	4	0
Smoking	7	1	0
Dyslipidemia	5	2	0

Abbreviations: AMI: acute myocardial infarction; CVR: cardiovascular risk; F: female; M: male.

Cell isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral venous blood by density gradient centrifugation with Ficoll-Hypaque (Sigma-Aldrich). Cellular viability was evaluated by staining with trypan blue. Before cell staining, cells were cultured in supplemented RPMI-1640 medium (10% fetal bovine serum, glutamine 10 nM, Streptomycin 100 μg/ml, penicillin 100 U/ml) with brefeldin-A (10 μg/ml, eBioscience) for 4 hours at 37°C, 5% CO₂.

Flow cytometry

Cell surface staining was performed with the following monoclonal anti-human antibodies labeled with different fluorochromes anti-: CD4-FITC, CD25-APC Cy7, CD69-APC, and LAP (TGF-β)-PerCp Cy5.5. Then, cells (PBMCs) were fixed and permeabilized with Foxp3 Fix/Perm kit (eBioscience) and stained with antibodies against IL-10 (conjugated with PE) and Foxp3 (PE Cy7). Cells were analyzed with the FACSCanto II flow cytometer (BD Biosciences, Becton Dickinson) and the FlowJo software (Tree Star, Ashland, OR). In all assays, FcR receptors were previously blocked with 10% human AB serum. We used a PE isotype control and applied the fluorescence minus one (FMO) strategy to set gates. In brief, FMO controls leave out one reagent (the one of interest) at a time (the opposite of single stain controls). In FMO, control is defined as changing one condition at a time.

Magnetic labeling and separation

CD4+ T cells were purified using MACS-MS columns (Miltenyi Biotec) by negative selection. In brief, PBMCs were incubated with a cocktail of biotin-conjugated antibodies against non-CD4⁺ T cells and later with anti-biotin MicroBeads (CD4⁺ T Cell Isolation Kit II human, MACS Miltenyi Biotec). A purified cell fraction was depleted of CD25⁺ cells with the CD25 MicroBeads II (human, MACS Miltenyi Biotec). Purified CD4⁺ CD25^- and CD4⁺ T cells were used to evaluate inhibition of cell activation.

Functional assay

To analyze the Treg cell suppressive function, we perform a cell activation inhibition assay. In this assay, CD4⁺ T cells and CD4⁺ CD25⁻ T cells (non-Treg) obtained from magnetic separation (MACS-MS columns, Miltenyi Biotec) were stimulated for activation by culturing in supplemented RPMI-1640 medium for 7 hours at 37°C with 5% CO₂ in precoated 48-well plates (Costar) with antibodies anti-CD3 (OKT3 clone, 10 μg/ml, BioLegend) and anti-CD28 (CD28.2 clone, BioLegend) for 1 hour at 37°C, 5% CO₂. At the time of beginning the cell culture, an anti-CD40L-PE antibody (Becton) was added, and after 7 hours of incubation, cells were harvested. Cells were acquired in FACSCalibur flow cytometer (BD Biosciences, Becton Dickinson), and CD40L fluorescence was analyzed using FlowJo software.

Soluble cytokine quantification

To measure soluble cytokine levels in serum from patients and control subjects, we performed a cytometric bead array (BD Biosciences), a flow cytometry application that simultaneously quantifies multiple proteins. Briefly, serum samples were incubated with capture beads and detector antibodies; then, sandwich complexes were formed with these reagents and the analyte. These complexes were measured using flow cytometry according to the characteristics of both the bead and the detector antibody.

Statistical analysis

Data were analyzed with GraphPad Prism, 5.01 software. Data were analyzed for normality with the Pearson test, Kolmogorov–Smirnov test, D’Agostino, and Shapiro–Wilk test. Flow cytometry data were evaluated by paired or unpaired Student t test in case of a normal distribution; otherwise, we used the Mann–Whitney U test or Wilcoxon signed-rank sum-test. In some cases, data were analyzed by one-way ANOVA, Friedman test, or two-way ANOVA test. The analysis of correlations between variables was based on Spearman’s rank test. A P value of <0.05 was considered statistically significant. The data are shown as mean and SD, or median with interquartile range (IQR).

Results

Analysis of CD4⁺ CD25^hi Foxp3⁺ IL-10⁺ T cells in peripheral blood from AMI patients

Circulating Treg cell levels were analyzed from the peripheral blood of AMI patients and controls. Conventional Treg cells were characterized by CD4, CD25 high, Foxp3, and IL-10 expression. Cell staining was performed as described in Material and Methods. Cells were analyzed by multiparametric flow cytometry following FMO strategy (Fig. 1A). First, we observed that patients with recent AMI (within 72 hours) showed increased levels of circulating CD4⁺ CD25^hi Foxp3⁺, compared to the control groups of healthy subjects (P = 0.0009, 0.5800%, 0.7300–0.4100% vs. 0.2550%, 0.3250–0.1675%, median and IQR, Fig. 1B) and those with CVR (P = 0.0547, 0.58%, 0.73–0.41% vs. 0.45%, 0.46–0.375%, median and IQR, Fig. 1C), but without previous myocardial infarction. IL-10 production by these Foxp3⁺ Treg cells at 72 hours after the ischemic event was higher than healthy subjects (P = 0.0108, 580.6 ± 127.5% vs. 443 ± 91.68%, arithmetic mean and SD, Fig. 1D).

Figure 1.

Analysis of CD4⁺ CD25^hi Foxp3⁺ IL-10⁺ Treg cells in peripheral blood from AMI patients and controls. (A) PBMCs were obtained by density gradient and then labeled with antibodies anti-CD4 and anti-CD25. For the staining of IL-10 and Foxp3, cells were fixed and permeabilized. Cells were analyzed by flow cytometry following fluorescence minus one (FMO) strategy. Percentages of conventional Treg cells populations referred to total lymphocytes, in peripheral blood from patients at 72 hours post-AMI, compared to healthy subjects (B, n = 19, 8), and subjects with CVR (C, n = 19, 5). (D) IL-10 median fluorescence intensity from CD4⁺ CD25^hi Foxp3⁺ cells in AMI patients within the first 72 hours (n = 19) compared with healthy subjects (n = 8). Data shown in panels (B) and (C) had a non-Gaussian distribution and correspond to the median and Q1 and Q3. Data shown in panel (D) had a normal distribution and correspond to the arithmetic mean and SD. Groups were compared using Mann–Whitney test or unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.

We also analyzed the frequencies of CD4⁺ CD25^hi Foxp3⁺ IL-10⁺ cells in patients who presented AMI within the first 72 hours as well as 3 and 6 months later. Interestingly, we found a significant decrease in the percentages of CD4⁺ CD25^hi Foxp3⁺ Treg cells between AMI within 72 hours and 6 months after AMI groups (P = 0.0099, Fig. 2A). Importantly, we observed a trend in the correlation analysis between Treg cell levels and percentage of LVEF 3 months after AMI; the higher levels of Treg cells were observed in subjects with a LVEF higher than 50% (P = 0.0717, Fig. 2B). However, in our study, most of the patients included presented LVEFs below 50% in the three sampling times. Regarding IL-10 production by these cells, we observed that this regulatory cytokine tended to increase 3 months after the ischemic event, diminishing after 6 months of AMI (P = 0.0375, Fig. 2C).

Figure 2.

Treg cells percentages over time. Comparison of CD4⁺ CD25^hi Foxp3⁺ Treg cells percentages referred to total lymphocytes (A, n = 8) and its IL-10 expression as median fluoresce intensity (C, n = 8) at 72 hours, 3 months, and 6 months after AMI. LVEF percentages correlation with CD4⁺ CD25^hi Foxp3⁺ cells frequency 3 months post-AMI (B, n = 8). All data shown had a non-Gaussian distribution. Groups were compared using one-way ANOVA (Friedman test). Spearman r analysis was performed. Linear regression was performed. P value represents the likelihood of a nonzero slope. *P < 0.05, **P < 0.01, ***P < 0.001.

Functional analysis of CD25⁺ Treg cells

To assess Treg cells suppressive capacity, we determined T-cell activation in the presence or absence of Treg cells in patients with AMI within 72 hours, 3 and 6 months postinfarction, and a control group with CVR without AMI. CD4⁺ T cells were obtained by magnetic separation, then were depleted or not of CD25⁺ T cells. Both fractions were stimulated with an anti-CD3 and anti-CD28 monoclonal antibody for 7 hours at 37°C. Then, the expression of CD40L was assessed by flow cytometry (Fig. 3A). The nondepleted fraction was expected to show mild CD40L expression, meaning mild activation, compared to CD25 depleted condition, which was expected to show higher activation since the regulatory cell population is absent. As expected, we found that in CVR control subjects, the expression of CD40L in the complete fraction of CD4⁺ cells was lower compared with the depleted fraction (P = 0.0313, 27.20%, 32.58–23.10% vs. 34.8%, 38.53–23.4%, median and IQR, Fig. 3B). We observed the same in AMI patients within the first 72 hours (P = 0.0431, 28.14 ± 12.42% vs. 34.35 ± 14.23%, arithmetic mean and SD, Fig. 3C). However, we did not observe changes in the percentages of CD40L⁺ cells between both fractions at 3 and 6 months after infarction (P = 0.0657 and P = 0.4688, Fig. 3D and E). Thus, these functional assays showed that CD25⁺ Treg cells seem to exert a suppressive effect on the activation of autologous T cells in controls with CVR and AMI patients at 72 hours but appear to be compromised in AMI patients in the late phases.

Figure 3.

Analysis of inhibition of lymphocyte activation by Treg cells CD25⁺ at 72 hours, 3 and 6 months post-AMI, and in a control group. CD4⁺ T cells were obtained by magnetic separation, then, were depleted or not from CD25⁺ T cells, both fractions were stimulated with antibodies versus CD3 and CD28 for 7 hours at 37°C. After incubation, the expression of CD40L was assessed. (A) Data of a representative experiment from a control subject are shown, and numbers correspond to the percent of CD40L⁺ cells. Analysis of inhibition of lymphocyte activation by Treg cells CD25⁺ in a control group of subjects with CVR (B, n = 6), in patients 72 hours post-AMI (C, n = 15), and 3 months (D, n = 9) and 6 months (E, n = 6) later. Data shown in panels (B) and (E) had non-Gaussian distribution and correspond to the median and Q1 and Q3. Data shown in (C) and (D) panels had normal distribution and correspond to the arithmetic mean and SD. Groups were compared using Wilcoxon test or paired t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Identification of CD4⁺ CD25^var CD69⁺ LAP⁺ Foxp3⁻ IL-10⁺ Treg cells in human peripheral blood from AMI patients at 72 hours after infarction, 3 and 6 months later

The recently described CD4⁺ CD69⁺ T-cell subset has been reported to exert an immunosuppressive effect. We decided to evaluate this population in blood samples from AMI patients, healthy subjects, and from subjects with CVR as control group. To identify CD4⁺ CD25^var CD69⁺ LAP⁺ Foxp3⁻ IL-10⁺ cells, we performed multiparametric flow cytometry as stated in Material and Methods (Fig. 4A). We observed similar levels of CD4⁺ CD25^var CD69⁺ LAP⁺ Foxp3^- IL-10⁺ Treg cells (referred to total lymphocytes) between the different groups studied. In patients with AMI (within 72 hours) and the healthy control group, no significant difference was found (P = 0.1265, 0.0250%, 0.0440–0.01175% vs. 0.0350%, 0.09625–0.0255%, median and IQR, Fig. 4B), or between these patients and the group with CVR (P = 0.9406, 0.0250%, 0.0440–0.01175% vs. 0.0240%, 0.1285–0.007325%, median and IQR, Fig. 4C).

Figure 4.

Analysis of CD4⁺ CD25^var CD69⁺ LAP⁺ Foxp3⁻ IL-10⁺ T cells in peripheral blood from AMI patients and controls. (A) PBMCs were obtained by density gradient and then labeled with antibodies versus CD4, CD25, CD69, and LAP (TGF-β). For IL-10 and Foxp3 staining, cells were fixed and permeabilized. Cells were analyzed by flow cytometry following fluorescence minus one (FMO) strategy. To evaluate the frequency of CD69⁺ Treg cells referred to total lymphocytes, cells were evaluated in peripheral blood from AMI patients (n = 18) within the first 72 hours compared to healthy (B) and CVR (C) controls (n = 8, 5), and from patients 6 months after the AMI (n = 8) versus healthy controls (E). Follow-up analysis of frequencies of CD69⁺ Treg in patients with AMI at 72 hours, 3 and 6 months later (D, n = 8). %LVEF relationship with CD69⁺ Treg frequency at 72 hours (F, n = 17) and 3 months post-AMI (G). All data had non-Gaussian distribution, the median and interquartile range are shown in panels (B), (C), and (E). Groups were compared using Mann–Whitney test, one-way (Friedman test), or two-way ANOVA. Spearman r analysis was performed. Linear regression was performed. P value represents the likelihood of a nonzero slope. *P < 0.05, **P < 0.01, ***P < 0.001.

Furthermore, when we evaluated possible changes in the percentage of CD4⁺ CD25^var CD69⁺ LAP⁺ Foxp3⁻ IL-10⁺ cells in patients with AMI at the different sampling times after infarction, we found no significant differences in the levels of these cells (P = 0.6543, Fig. 4D). It is noteworthy, that 6 months after the AMI, the CD69+ Treg cell levels were lower than these observed in healthy subjects (P = 0.0224, 0.0165%, 0.03475–0.01425% vs 0.0350%, 0.09625–0.0255%, median and IQR, Fig. 4E). Nevertheless, we could not find a significant correlation between the levels of CD69⁺ Treg cells and the percentage of LVEF of patients within 72 hours postinfarction (P = 0.4001, Fig. 4F). In most of the AMI patients included, CD69⁺ Treg cell levels increased at 3 months after infarction independently of the LVEF. Only one patient showed a decrease in CD69⁺ Treg cells, it is important to point out that no significant clinical features were identified in this patient (Fig. 4G).

Measurement of soluble cytokine levels in serum from patients and control subjects

Inflammatory cytokines play an important role in the outcome of AMI patients. We evaluated the serum concentration of IL-6, IL-1β, and IL-8 from patients within 72 hours after the AMI. We found a negative correlation between IL-6 levels (pg/ml) and CD4⁺ CD25^hi Foxp3⁺ frequency, indicating that there is an association between a high percentage of Treg cells and a lower concentration of serum IL-6 (P = 0.0091, Spearman r = −0.8675, Fig. 5A). IL-1β serum levels did not show differences at 72 hours and 3 months after AMI; however, we noticed a downward trend at 6 months after the acute event (Fig. 5B, P = 0.1111). Regarding the levels of IL-8, we did not find any difference at the three sampling times (within 72 hours, 3 and 6 months after the infarction; P = 0.6528; Fig. 5C).

Figure 5.

Analysis of serum cytokine concentration from patients with AMI. (A) Association among the percentages of CD4⁺ CD25^hi Foxp3⁺ cells and IL-6 concentration (pg/ml) from AMI patients’ serum within the first 72 hours after the event (n = 8). IL-1β (B, n = 5) and IL-8 (C, n = 4) levels (pg/ml) in patients who suffered AMI within the first 72 hours, 3 and 6 months later. All data shown had non-Gaussian distribution. Groups were compared using one-way ANOVA (Friedman test). Spearman r analysis was performed. Linear regression was performed. P value represents the likelihood of a nonzero slope. *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

Treg cells are cells with the ability to inhibit autologous cell activation and proliferation, mainly effector T lymphocytes [21]. These lymphocytes were first described by Sakaguchi et al. [7] over 20 years ago, and from that moment, Treg cells have become the focus of many studies because of their essential role in regulating the immune response to maintain homeostasis and prevent autoimmunity.

Treg cell suppressor capacity is mediated by cytokines or in a cell–cell contact-dependent manner [9]. Several subpopulations of Treg cells have been characterized by the expression of different molecules. Treg cells can be classified according to their origin in thymic Treg cells (tTreg), differentiated in the thymus and, in pTreg, which are induced in the periphery; both populations are CD4⁺ CD25^hi and Foxp3⁺ [22]. According to Foxp3 expression, there are subpopulations of Treg cells Foxp3⁻, such as CD4⁺ CD25⁻ Foxp3⁻ lymphocytes, which exert their suppressive function through the release of IL-10, termed Tr1 [23], or through the release of TGF-β, termed Th3 [22].

The role of conventional Treg cells (CD4⁺ CD25^hi Foxp3⁺) in CVD has been widely described. In atherosclerosis, Treg cells have a beneficial role [24] and, in ACS such as AMI, these cells are diminished [8]. A recent study in infracted mice shows that an increased frequency of these cells and their production of IFN-γ lead to a poor outcome [17].

We evaluated the conventional Treg cell population characterized by the expression of CD4, CD25 high and Foxp3 in peripheral blood from AMI patients within the first 72 hours. The percentages of these Treg cells (CD4, CD25 high, and Foxp3) in patients who suffered AMI are higher than in subjects who did not suffer an infarction, whether healthy or with CVR factors. Treg cells Foxp3⁺ IL-10⁺ are essential contributors to immune homeostasis in the lungs [25], large and small intestine [26], but their role in AMI remains unclear. We demonstrated the production of IL-10 by these Foxp3⁺ Treg cells after the ischemic event and the expression of this inhibitory cytokine is higher in these patients than in healthy subjects. Furthermore, their suppressive function was conserved within the first 72 hours, including the inflammatory phase. The increase in CD4⁺ CD25^hi Foxp3⁺ cells may indicate that a compensation mechanism is developed in response to the ischemia in the myocardium. This regulatory event may counter the postischemic acute inflammatory response. Furthermore, our results showing an increase in the percentage of Treg cells in circulation immediately after the infarction, accompanied by a lower concentration of serum IL-6 support the idea that Treg cells play a role in the control of the inflammatory process after AMI.

Previous studies have reported that Treg cell levels in patients with AMI are diminished [27], although it has been shown in mice that there is an activation of these cells immediately after the ischemic event [5]. Furthermore, in patients with established heart failure, lower percentages of Treg cells, and an impaired function have also been found compared to healthy controls [20]. For that reason, we also analyzed the frequency of CD4⁺ CD25^hi Foxp3⁺ cells and their IL-10 production in patients 3 and 6 months later after AMI. We proved that 6 months after the post-AMI inflammatory phase occurred, there was a decrease in the frequency of this cell population and its IL-10 expression.

CD40L is a molecule that is expressed in a transitory manner in activated T lymphocytes [28]. Functional activation assays showed that in the absence of CD25⁺ cells (fraction in which Treg cells are included), T effector cells enhance their activation because there is no one to regulate or limit the activation of the effector cells, so the expression of CD40L is higher compared to the complete fraction of CD4⁺ cells, containing effector T lymphocytes and Treg cells. This was true for our CVR control group studied. Treg cell suppressive function is also maintained immediately after the infarction (at 72 hours), having a behavior similar to Treg cell suppressive function in CVR control subjects.

When we analyzed CD25⁺ Treg cell function 3 and 6 months after AMI, we found that its immunomodulatory function was affected since there is no difference in the degree of activation of T lymphocytes (% CD40L⁺ cells) when CD4⁺ CD25⁺ Treg cells are present or absent. This may indicate that although this regulatory subset is present, its function is compromised since they do not exert the necessary suppressive function to inhibit the activation of effector T lymphocytes [8]. In previous studies, it has been found that both, function and frequency, of Treg cells are decreased in AMI patients [19] as well as in patients with heart failure [20].

Nevertheless, the behavior of these cells over time has never been studied. In this study, we found that since infarction onset, besides the frequency, Treg cell function fails at least 3 and 3 months later. This phenomenon could be related to ventricular failure since most of our patients have decreased left ventricular functionality. Interestingly, we found that at least 3 months after the infarction, there is a trend of an association between a higher percentage of Treg cells and a better preserved LVEF, even when the Treg cell inhibitory function is compromised. Our findings are in accordance with Bansal et al. [17] who demonstrated in murine models that dysfunctional Treg cells are essential for adverse cardiac remodeling.

As mentioned above, different populations of Treg cells have been described. CD4⁺ CD25⁻ CD69⁺ Foxp3^- Treg cells were first described in mice [14]. In humans, it has been shown an increase in the expression of CD69 in peripheral blood cells CD4⁺ CD25⁺ Foxp3⁺ after AMI, and it was associated with a lower risk of rehospitalization for heart failure [29], highlighting the importance of evaluating the different populations of CD69+ Treg cells in scenarios of ischemia such as AMI. Furthermore, a population of CD4⁺ CD69⁺ LAP (TGF-β)⁺ Foxp3⁻ has also been described. This population differentiation is independent of the Foxp3 transcription factor. These cells also express the molecule CD25 variably, and besides, its suppressor function is mediated by IL-10 and TGF-β secretion [30]. Zhu et al. [11] evaluated CD4⁺ LAP⁺ Treg cells in ACS patients, they found lower frequencies and a diminished function of these cells compared to controls (patients with chronic stable angina and chest pain syndrome), but CD4⁺ CD25^var CD69⁺ LAP⁺ Foxp3⁻ IL-10⁺ in AMI patients has not been evaluated until now to our best knowledge.

In patients suffering from AMI, within the inflammatory phase, which comprises the first 72 hours, we provide evidence that there is no significant difference in the percentages of CD69⁺ Treg cells (CD4⁺ CD25^var CD69⁺ LAP⁺ Foxp3⁻ IL-10⁺) when compared with the percentages of these cells in healthy subjects. After the infarction inflammatory phase, there is a phase of the lesion’s resolution in the myocardium. Through the change of these phases, we did not find significant variations in the percentages of circulating CD69⁺ Treg cells, nor a relationship between the circulating levels of these cells and the ventricular function evaluated with the percentage of LVEF. It would be interesting to evaluate if there is a fluctuation in the amount of these cells present in the myocardial tissue. Interestingly, 6 months after the patients suffered the infarction, we found that these Treg cells that constitutively express CD69 are low if we compare them with the percentages found in people who had not suffered this acute event or presented any type of CVD. This difference only becomes noticeable up to six months, not at 72 hours or 3 months. However, the relevance of this observation remains to be explained, since we could not find any correlation between the frequency of this nonconventional Treg subset and any cardiac function parameter. It would be important to perform further studies to evaluate the suppressive function of this subset in acute and chronic cardiac pathologies. The frequencies of this subset in our studied groups are in agreement with the data of Vitales et al. [15] who reported a frequency of 0.026% of CD4⁺ CD25^var CD69⁺ LAP⁺ Foxp3⁻ IL-10⁺ cells referred to total lymphocytes in peripheral blood of healthy subjects. In this regard, it is important to mention that the frequencies of conventional CD4⁺ CD25^hi Foxp3⁺ populations found in our study are below the described values in healthy subjects, which are ranging from 5% to 10% of total CD4⁺ T cells [31]. This may be explained by genetic characteristics owned by our population studied, emotional or physical stress [32], or due to cardiovascular factors we did not assessed, such as lipid levels.

In conclusion, this is the first known follow-up study evaluating CD69⁺ Treg cells with the phenotype CD4⁺ CD25^var CD69⁺ LAP⁺ Foxp3⁻ IL-10⁺ in AMI patients as well as conventional Treg cells CD4⁺ CD25^hi Foxp3⁺ IL-10⁺, 3 and 6 months after AMI. CD4⁺ CD25^high Foxp3⁺ Treg cells and their IL-10 production play an important role in regulating inflammation after an AMI, and its function may be compromised in this pathology from the AMI onset when there is ventricular dysfunction. This population may play an essential role in limiting inflammation in myocardial infarction and different systemic inflammatory diseases. However, further studies must be done in order to determine the physiological relevance of CD69⁺ Treg cells CD4⁺ CD25^var CD69⁺ LAP⁺ Foxp3⁻ IL-10⁺ in maintaining cardiac function because its involvement in the metabolic reprogramming regulating T helper lymphocyte lineages.

Glossary

Abbreviations

APC

antigen-presenting cells

ACS

acute coronary syndrome

AMI

acute myocardial infarction

CVD

cardiovascular diseases

CVR

cardiovascular risk

FMO

fluorescence minus one

IQR

interquartile range

LAP

latent associated peptide

LVEF

left ventricular ejection fraction

nTreg

natural Treg cells

PBMCs

peripheral blood mononuclear cells

pTreg

peripheral Treg cells

Treg

regulatory T

SD

standard deviation

Tr1

T regulatory 1 cells

tTreg

thymic Treg cells

TGF-β

transforming growth factor-β.

Ethical approval

Local University Ethics Committee (UASLP) approved this study (approval number 58-14). This work was carried out following The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.

Conflict of interests

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by CONACYT with the financial support of ‘Proyecto de Conacyt Ciencia Básica’ [CB-2012-01 No.180094 (A.E.M.U.)].

Data availability

The data underlying this article are available in the article and Supplementary material.