A Unique Population of Regulatory T Cells in Heart Potentiates Cardiac Protection From Myocardial Infarction

Introduction

Regulatory T cells (Tregs), especially the subset of CD4⁺Foxp3⁺ Tregs, are critical in maintaining immune homeostasis and regulating inflammatory disease progressions.¹ Tregs can modulate the activation and function of a variety of immunocytes, including innate and adaptive immune system players.² In addition to their immunoregulatory function, existing evidence shows that Tregs have extraimmunologic roles, for example, promoting effective tissue repair in peripheral tissues.³ The initial research on tissue-resident Tregs came from the Mathis team that described the characteristics of visceral adipose tissue Tregs and injured muscle Tregs.^4–6 Visceral adipose tissue Tregs highly express peroxisome proliferator–activated receptor-gamma and enhance glucose metabolism in adipocytes, which mitigates insulin resistance.^4,5 However, injured muscle Tregs express Areg (amphiregulin), targeting the muscle satellite cells and maintaining tissue integrity.⁶ Since then, Tregs with unique phenotypes and functions have been reported in the pancreas,⁷ skin,⁸ brain,⁹ and lung.¹⁰ Tregs in the hearts, especially undergoing myocardial infarction (MI), however, remain largely undefined.

MI is one of the most common cardiovascular diseases, with ventricular rupture and heart failure as its major causes of death. Therefore, preventing cardiac rupture and controlling ventricular remodeling in the early and late stages, respectively, can effectively improve clinical outcomes.¹¹ A variety of immune components, including Tregs, are involved in the repair after MI.^12–14 Elimination and expansion experiments confirmed that Tregs play a protective role in MI by alleviating local inflammation, protecting cardiomyocytes from apoptosis, and modulating macrophage differentiation and myofibroblast activation.^12–14 However, these findings leave open the question: What is the characteristic of Tregs themselves in MI hearts? Specifically, whether Tregs have local adaption with unique phenotype, how the populations accumulate in MI hearts and what factors drive their accumulation are largely unknown. It is most important to note that whether they were endowed with unique functions in hearts such as directly targeting the cardiac repair process independent of immune regulation remains to be fully elucidated.

In the present study, we set up to investigate the characteristic of Tregs accumulated in MI hearts through multiple transcriptional and functional assays. The results revealed a unique population of tissue-related Tregs in the hearts after MI, characterized their distinct phenotypes, sources, and sustaining factors, and support the emerging role of Tregs in cardiac repair. Our study therefore provided insight into developing new therapy for the clinical treatment of MI by using Tregs.

Methods

On reasonable request, the data that support the findings of this study are available from the corresponding author. Extended methods are provided in the Data Supplement.

Animals

All mice used in our study were housed under specific pathogen-free conditions. All animal studies were conducted according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985) and approved by the Animal Care and Utilization Committee of Huazhong University of Science and Technology, China. The procedures followed were in accordance with institutional guidelines.

Reverse Transcription Polymerase Chain Reaction

Total RNA was extracted using TRIzol reagent, and mRNA levels were quantified by reverse transcription polymerase chain reaction (RT-PCR). The primers used for RT-PCR are listed in Table I in the Data Supplement.

Statistical Analysis

Data are presented as the mean±SEM. The Kolmogorov-Smirnov test with Lilliefors correction was first used to determine data normality, and the F test was used to determine equal variance. If the data passed the evaluation of normality and similar variances, statistical significance was determined by 1- or 2-way ANOVA with Tukey post hoc multiple comparisons tests to analyze differences among ≥3 groups, or by unpaired Student t test to analyze the differences between 2 groups. The nonparametric tests, such as Kruskal-Wallis test, Scheirer-Ray-Hare test, or Mann-Whitney U test were used when the data displayed abnormal distribution, and these data were presented in the form of box-and-whisker plots to show the median and ranges. Survival curves were obtained by the Kaplan-Meier method and compared by the log-rank test. In all cases, a 2-tailed P value of <0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 8.0.

Results

Tregs Are Accumulated in the Injured Myocardium

To monitor the infiltration of heart Tregs at various times after MI, the dynamic analysis was performed by flow cytometry. The gating strategy of Tregs in hearts is shown in Figure 1A. Tregs in the sham-operated hearts, spleens, or peripheral blood were evaluated separately. Tregs in myocardium had a tendency to increase at day 1 post-MI, reached the highest value at day 7, and still maintained increased values at day 14 (Figure IA in the Data Supplement). The number of heart CD4⁺ T cells and heart conventional T cells (Tconvs) both peaked at day 7 after MI, parallel to heart Tregs (Figure IB and IC in the Data Supplement). The frequency of heart Tregs within the CD4⁺ T cells gradually increased 7 days after MI, and maintained values until at least 28 days (Figure 1B). In contrast, the proportion of Tregs in the spleen and blood showed little variability over the time course (Figure 1B).

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A similar accumulation of Tregs was also observed in hearts after myocardial ischemia/reperfusion injury (I/R injury). Tregs infiltrated into injured hearts within 1 day post-I/R injury, then peaked at day 3 and remained elevated until 7 days (Figure ID in the Data Supplement). The frequency of heart Tregs also reached a peak at day 3 after I/R injury, which was higher than that in blood and spleens (Figure 1C). In addition, the number of Tregs was also found increased in cryoinjured hearts. Tregs accumulated in the cryoinjured myocardium 1 day after injury and peaked at day 5 (Figure 1D). Taken together, these results indicate a possible role of heart Tregs after cardiac injury.

Transcriptome of Heart Tregs Has Unique Characteristics

To identify the transcriptional phenotype of heart Tregs, Tregs and Tconvs were isolated from hearts, spleens, and nondraining lymph nodes (NDLNs) 7 days after MI to perform RNA sequencing. Volcano plots revealed that the transcriptome of heart Tregs apparently differed from spleen or NDLN Tregs, whereas the latter 2 shared more similarities (Figure 2A). The differences between Tregs in hearts and lymphoid organs cannot be simply explained by different activation states in nonlymphoid tissue, because Treg activation signature^6,15 only accounted for a very small fraction of the distinguishing transcripts (Figure 2B and Figure IIA in the Data Supplement). Although heart Tregs exhibited a distinct transcriptome, they were clearly Tregs based on the expression of 79% of the canonical Treg signature,^5,6 including elevated expression of hallmark transcripts Foxp3, CD25, CTLA-4, and Klrg1 (Figure 2C).

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Some upregulated differentially expressed genes in skin⁸ and muscle¹⁶ Tregs were also augmented in heart Tregs (Figure IIB in the Data Supplement). However, principal components analysis and transcriptomic analysis suggested transcriptional differences among these 3 tissue Tregs and between tissue Tregs and lymphoid organ Tregs^8,16 (Figure 2D and Figure IIB in the Data Supplement). Then we analyzed those upregulated differentially expressed genes in heart Tregs. The first group included Ctla4, Areg, and Il1rl1, which were also upregulated in muscle Tregs and skin Tregs. The second group was numerous cytokines, cytokine receptors, chemokines, and chemokine receptor–related genes (eg, Il10, Il4ra, Ifngr1, Irf1, Tnf, Tnfrsf9, Ccr2, Ccr7, Ccr8, Ccrl2, and Cxcl10). It is notable that a group of extracellular matrix organization– or collagen synthesis–related genes (eg, Sparc, Dcn, Bgn, Mgp, Serpinh1, Postn, Col1a1, Col3a1, and Fn1) were strikingly upregulated in heart Tregs (Figure 2E). Gene set enrichment analysis demonstrated that genes upregulated in heart Tregs displayed enrichment for Gene Ontology biological process terms involved in extracellular structure organization, extracellular matrix organization, collagen metabolic process, collagen biosynthetic process, wound healing, and regulation of smooth muscle cell proliferation, which were related to tissue repair (Figure 2F, Tables II and III in the Data Supplement).

To address whether the altered genetic signatures in heart Tregs are attributable to in situ microenvironment of cardiac injury, we compared the transcriptome of heart Tregs with heart-draining mediastinal lymph node (MLN) Tregs 7 days after MI. Volcano plots and principal components analysis revealed that the transcriptome of heart Tregs also apparently differed from MLN Tregs (Figure IIC in the Data Supplement and Figure 2D). The upregulated genes of heart Tregs and Gene Ontology biological process terms of heart Tregs mentioned earlier also significantly upregulated in heart Tregs in comparison with MLN Tregs (Figure 2E, Figure IIB and IID in the Data Supplement).

To identify the transcriptional characteristics of heart Tregs in I/R injury, Tregs were isolated from hearts, spleens, and MLNs 3 days after I/R injury to perform RNA sequencing. Principal components analysis and volcano plots showed that the transcriptome of heart Tregs significantly differed from spleen or MLN Tregs (Figure 2D, Figure IIE and IIG in the Data Supplement). Gene set enrichment analysis showed that genes involved in extracellular matrix organization and extracellular structure organization, which are related to tissue repair, were significantly upregulated in heart Tregs in comparison with spleen or MLN Tregs (Figure IIF and IIH in the Data Supplement).Through principal components analysis and transcriptomic analysis, we found that heart Tregs from 2 models shared very similar transcriptome (Figure 2D and Figure IIB in the Data Supplement). In summary, our data suggest heart Tregs are a novel population of tissue Tregs, potentially involved in cardiac repair.

Heart Tregs Are Mainly Derived From the Circulating Compartment After MI

To identify the sources of heart Tregs after MI, we first asked whether the circulating Treg pool contributed to the accumulation of heart Tregs. Mice were treated with FTY720, an S1P1 receptor agonist that retains lymphocytes in secondary lymphoid tissue.¹⁷ Under the treatment with FTY720, both the numbers and proportions of Tregs recruited to ischemic hearts markedly dropped on day 3 and day 7 after MI, whereas the number of CD4⁺T cells infiltrating into infarcted hearts also showed a significant decrease (Figure 3A). These results indicate that the accumulation of heart Tregs after MI may depend on the circulating Treg pool.

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Therefore, a series of experiments were conducted to evaluate the ability of Tregs to infiltrate into the infarcted myocardium from circulation after MI. First, the kikGR/B6-ROSA transgenic mice were used to track the migration of Tregs and Tconvs. We exposed cervical lymph nodes of kikGR mice to violet light 3 days after MI, and tracked the Kikume-red⁺ Tregs and Tconvs from the cervical lymph nodes to the axillary lymph nodes, MLNs, and hearts 4 days after photoconversion (Figure 3B). Kikume-red⁺ Tregs and Tconvs emigrated from the cervical lymph nodes in mice, and the frequency of photo-converted cells within the Tregs or Tconvs were measured in the hearts, MLNs, and axillary lymph nodes (Figure 3C and 3D and Figure IIIA in the Data Supplement). When the fractions of Kikume-red⁺ cells in hearts and MLNs were normalized to that in axillary lymph nodes to calculate a migration ratio, Tregs manifested more effective migration to ischemic hearts than Tconvs, whereas no significant difference in migration between Tregs and Tconvs was observed in MLNs (Figure 3E).

Next, for detailed analysis of the contribution of the circulating Treg pool to heart Tregs, we performed a parabiosis experiment. We conjoined CD45.1 and CD45.2 mice for 2 weeks to establish a shared circulation.¹⁸ Then, coronary ligation was performed on the CD45.1 parabiont, and the chimerism of Tregs was examined 7 days post-MI (Figure 3F and 3G). Chimerism of the heart Tregs was comparable to the admixture of the blood Tregs (Figure 3H, Left). Based on these data, we estimated that the recruitment of Tregs from the circulation accounted for nearly 95% of the Tregs within the infarcted hearts (Figure 3H, Right, and Figure IIIB in the Data Supplement). Our results collectively suggest that the accumulation of Tregs in the post-MI hearts mainly depends on recruitment from the circulating Treg pool.

Heart Tregs Are Clonally Expanded That Display a Unique T-Cell Receptor Repertoire

In addition to influx from the circulation, proliferation may contribute to the rapid accumulation of heart Tregs after MI. To address this possibility, we stained Tregs with Ki67. We found that ≈75% of heart Tregs 7 days after MI were Ki67-positive, much higher than those in spleen or NDLN Tregs (Figure 4A). In addition, we performed a proliferation assay by using 5-ethynyl-2′-deoxyuridine (EdU) incorporation. We detected ≈25% EdU⁺ cells among heart Tregs, but a lower fraction within spleen or NDLN Tregs (Figure 4B). The fractions of Ki67⁺ and EdU⁺ cells within heart Tconvs were also increased in comparison with lymphoid organ Tconvs, but lower than those in heart Tregs (Figure 4A and 4B). These findings indicate a higher proliferation rate of heart Tregs than Tconvs and lymphoid organ Tregs post-MI.

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To identify the clonality of the Tregs expanding in hearts post-MI, we applied paired single-cell RNA and T-cell receptor (TCR) sequencing of CD4⁺ T cells from 7-day post-MI hearts and paired spleens to study their TCR repertoires. Unsupervised clustering was performed by Seurat,^19,20 and the t-distributed stochastic neighbor-embedding approach²¹ was used to visualize clustering maps of heart and spleen CD4⁺ T cells (Figure 4C and 4D). The clusters of heart Tregs and spleen Tregs were identified by expression of Foxp3 (Figure 4C and 4D, Figure IVA and IVB in the Data Supplement). The characteristics of clonal restriction in heart Tregs were investigated by analyzing the TCR repertoires. A considerable fraction of heart Tregs was clonally expanded (Figure 4E, Left, and Figure IVC in the Data Supplement). In contrast, the spleen Tregs showed a much lower frequency of clonal expansion (Figure 4E, Right), and there were just a few TCR sequences shared between spleen Tregs and heart Tregs (Figure 4F). These results prove a unique TCR repertoire of heart Tregs and their local clonal expansion in the infarcted hearts after MI.

Conversion of Tconvs Makes a Small Contribution to Heart Treg Pool

Previous studies have shown that thymus-derived Tregs made up the majority of Tregs in secondary lymphoid organs. Some Tconvs can gain stable Foxp3 expression in the peripheral sites and can be converted to peripheral Tregs (pTregs).^22,23

First, we examined the expression of Helios and Nrp-1 (neuropilin 1) on heart Tregs 7 days post-MI, because these markers are considered to distinguish between thymus-derived Tregs and pTregs.^23,24 Most heart Tregs showed a thymic origin with the Helios^hiNrp-1^hi phenotype, similar to spleen or NDLN Tregs, indicating that the heart Tregs were mainly thymus derived (Figure 5A).

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To directly identify whether Tconvs can be converted to heart Tregs post-MI, CD45.2⁺ Tconvs were isolated and adoptively transferred into CD45.1 mice soon after MI. Donor-derived Tconvs (CD45.2⁺CD4⁺Foxp3^–) could be detected in organs 2 weeks after Tconvs adoptive transfer (Figure VA in the Data Supplement). It is surprising that a small population of donor-derived pTregs (CD45.2⁺CD4⁺Foxp3⁺) was detected not only in the lymphoid organs, but also in the infarcted hearts. Moreover, donor-derived pTregs showed an even higher frequency in the infarcted hearts than in the spleens and NDLNs (Figure 5B).

Finally, we analyzed the TCR repertoires of heart Tconvs after MI. When comparing the TCR repertoires of heart Tregs and heart Tconvs, identical TCR clonotypes were found, indicating a probable contribution of the conversion of Tconvs to the heart Tregs (Figure 5C and Figure VB in the Data Supplement).

Interleukin-33/ST2 Axis Governs Accumulation and Expansion of Heart Tregs

Next, we set up to determine the mechanism of heart Treg accumulation after MI. We noticed that Il1r1l, which encodes ST2 (suppression of tumorigenicity 2), the receptor of interleukin-33 (IL-33), was strongly upregulated in heart Tregs, and the increased transcript level was further confirmed by RT-PCR (Figures 2E and 6A and 6B). Considering that IL-33 is related to Treg homeostasis in parenchymal tissues,^16,25,26 we hypothesized that heart Tregs might accumulate and expand in response to IL-33.

Flow cytometry was performed to measure ST2 protein. The percentage of ST2⁺ Tregs within heart Tregs was much higher than spleen Tregs, and the fraction of ST2⁺ heart Tregs elevated at day 3 and sustained until at least 28 days (Figure 6C and Figure VIA in the Data Supplement). Moreover, we found that both Il33 transcript and IL-33 protein levels were increased after MI, peaking at day 5 (Figure 6D and 6E). To pinpoint the cellular source of IL-33 in the infarcted hearts, we sorted the CD45⁺ and CD45^– cell fractions from the hearts 5 days post-MI and quantified their Il33 transcript levels, and found that IL-33 was mainly expressed by CD45^- cells (Figure VIB in the Data Supplement). Next, we sorted endothelial cells, cardiomyocytes, and cardiac fibroblasts from the post-MI hearts and quantified their Il33 transcript levels. Results showed that IL-33 was highly expressed in cardiac fibroblasts, and cardiomyocytes also expressed a small amount of IL-33, but endothelial cells hardly showed any expression (Figure 6F). Then immunofluorescence staining was used to confirm the cell source of IL-33, which indicated that the majority of IL-33–positive cells was Vimentin⁺ fibroblasts, except in rare cases were α-actinin⁺ cardiomyocytes, but none of them were costained with CD31, suggesting that the main source was cardiac fibroblasts, which was consistent with a previous study²⁷ (Figure 6G, Figure VIC and VID in the Data Supplement).

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Then we performed a series of loss- and gain-of-function experiments to evaluate the function of the IL-33/ST2 axis in the accumulation of heart Tregs after MI. The number of heart Tregs was markedly reduced in Il1rl1^–/– mice in comparison with wild-type mice 7 days after MI, whereas there were no obvious changes in spleen Tregs (Figure 6H). In contrast, administration of recombinant IL-33 induced an impressive expansion of heart Tregs 7 days post-MI, because the numbers and proportions of heart Tregs were markedly increased in IL-33–treated mice (Figure 6I and Figure VIE in the Data Supplement). IL-33 administration also elevated the proportions of proliferating heart Tregs (Ki67⁺CD4⁺Foxp3⁺) and ST2-positive heart Tregs (ST2⁺CD4⁺Foxp3⁺; Figure VIF in the Data Supplement). Besides, the spleen Tregs were also expanded and expressed ST2 and Ki67 more often under IL-33 treatment relative to phosphate-buffered saline treatment (Figure 6I and Figure VIF in the Data Supplement). These observations suggest that the IL-33/ST2 axis induces the heart Treg expansion by promoting their proliferation.

Heart Tregs Overexpressing Sparc Ameliorate Survival and Protect Against Rupture After MI

Previous studies suggest a reparative role of Tregs in myocardial healing after MI.^12–14 Tregs indeed perform anti-inflammatory activities after MI, whereas their tissue-distinct capacity to directly influence pathological processes within the infarcted hearts has not been fully elucidated.

In an attempt to further delineate the mechanisms by which heart Tregs promote tissue repair after MI, we analyzed the transcriptome of heart Tregs and noticed that Sparc was among the highest differentially expressed genes of heart Tregs (Figure 7A and Figure VIIA in the Data Supplement). Sparc (secreted acidic cysteine-rich glycoprotein, also known as osteonectin), a collagen-binding matricellular protein, plays a pivotal role in collagen assembly into the extracellular matrix and has been reported to play a crucial role in preserving ventricular integrity after MI.²⁸ The higher transcript level of Sparc in heart Tregs was further confirmed by RT-PCR (Figure 7B). Moreover, IL-33 treatment could induce upregulation of Sparc expression in heart Tregs (Figure 7C).

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To functionally determine whether Sparc plays a role in heart Treg-mediated tissue repair post-MI, we applied anti-CD25 antibody to deplete Tregs and administered a replication-deficient adenovirus harboring mouse Sparc (Ad-Sparc) to overexpress Sparc globally. Control adenovirus (Ad-Ctrl) was used as control (Figure VIIB in the Data Supplement). In the anti-CD25–treated groups, CD4⁺CD25⁺Foxp3⁺ Tregs presented a significantly reduced fraction in peripheral blood relative to the isotype control antibody immunoglobulin (Ig) G–treated group (Figure VIIC in the Data Supplement). Overexpression of Sparc in the infarcted hearts was identified 7 days and 14 days post-MI in the Ad-Sparc administration groups in comparison with Ad-Ctrl groups (Figure VIID in the Data Supplement). In comparison with Ad-Ctrl–treated Treg-ablated mice, the Ad-Sparc–treated Treg-ablated mice had significantly improved survival, attributing to fewer cardiac ruptures after MI (Figure 7D and 7E). In contrast, a difference in survival was not observed between Ad-Sparc–treated Treg-ablated mice and IgG+Ad-Ctrl–treated mice, indicating a near-complete rescue by Ad-Sparc (Figure 7D and 7E). Echocardiography was performed 14 days post-MI in surviving mice, and there were no statistically significant differences among those treatment groups (Figure 7F). No improvement of cardiac function was found after Sparc overexpression, which could presumably be explained by better survival of mice with poor cardiac function in the Ad-Sparc–treated group than in the Ad-Ctrl–treated group.

We next focused on scar formation of the infarct zone. Although the scar size did not significantly differ among these treatments (Figure VIIIA and VIIIB in the Data Supplement), the infarct thickness was increased in Ad-Sparc–treated Treg-ablated mice in comparison with that in the Ad-Ctrl–treated Treg-ablated group, which was consistent with the result of lower cardiac ruptures because a thick scar was not prone to rupture²⁹ (Figure VIIIA and VIIIC in the Data Supplement). Because collagen is the most abundant structural component of infarct scar tissue, the collagen content and maturation were analyzed after MI. First, Treg depletion reduced the Sirius red–stained collagen content of the infarct zone, and collagen III, as well, but not collagen I expression, whereas Sparc overexpression reversed these effects (Figure VIIID in the Data Supplement and Figure 8A through 8D). Because myofibroblast was the main producer of collagen in hearts after MI,^30,31 we further assessed the α-smooth muscle actin expression, but found no difference among these groups (Figure 8B). Next, as for collagen maturation, Sirius red polarization microscopy revealed a predominance of thicker tightly packed (orange-red) collagen fibers within infarct scars in Ad-Sparc–treated Treg-ablated mice or isotype control IgG-treated mice than in control Ad-Ctrl–treated Treg-ablated mice, indicating a remedy of collagen maturation by Sparc overexpression after Treg depletion (Figure 8E). Moreover, analysis of the collagen maturation by electron microscopy revealed that Treg depletion resulted in loose, disturbed, and thinner collagen fibers in the infarct scars, whereas collagen fiber organization was tight and parallel, and the diameter of collagen fibers was larger after Sparc overexpression, which also indicated improved collagen maturation (Figure 8F and Figure VIIIE in the Data Supplement).

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To further validate that Sparc was one of the important functional molecules in heart Tregs, we conducted in vitro experiments. Because tissue Tregs can hardly survive in vitro as previously described,⁵ we sorted spleen Tregs and overexpressed Sparc by Sparc-overexpressing lentivirus, and the overexpression was confirmed by RT-PCR (Figure VIIIF in the Data Supplement). We cocultured the cardiac fibroblasts with Tregs and found that cardiac fibroblasts that were cocultured with Sparc-overexpressing Tregs expressed a higher level of Col3a1 than cardiac fibroblasts that were cocultured with control Tregs or cultured alone (Figure 8G). In contrast, Col1a1 and ASMA did not show obvious differences (Figure 8G). Taken together, heart Tregs lead to increased collagen content and enhanced maturation in the infarct scars, and Sparc is critical in this process.

Discussion

Emerging evidence has recently established that tissue Tregs have multiple functions beyond their initial immunosuppressive effect within a variety of tissues.^3–10 Here, we identified that Tregs accumulated in infarcted hearts, with a distinctive transcriptome, represented a unique population of tissue Tregs. Heart Tregs were mainly recruited from circulation, whereas local expansion and conversion also contributed to the compartment. The IL-33/ST2 axis was important for expanding heart Tregs. Moreover, we focused on their direct ability of wound repair as suggested by transcriptomic data and identified Sparc as a functional mediator of heart Tregs to promote repair.

Tregs That Accumulate in the Injured Myocardium Have a Distinct Transcriptome

Based on the unique transcriptomic characteristics, we identified a previously unrecognized tissue-Treg population in hearts. The markedly altered transcripts in our transcriptome data, which first caught our attention, are genes encoding molecules related to Treg suppressive capacity. Ctla-4 and Klrg-1 were upregulated on heart Tregs, supporting the notion that heart Tregs may be endowed with especially strong inhibitory activity in the inflammatory microenvironment. More importantly, heart Tregs overexpressed extracellular matrix organization or collagen synthesis–related genes, distinguishing them from the lymphoid organ Tregs, whose products help them perform important functions in maintaining cardiac function and integrity after MI. Although the transcriptional profile has a certain degree of similarity, tissue Tregs are also highly complex and heterogeneous in different organs. These differences suggest that the microenvironment related to damage and specific tissues is critical in orchestrating Tregs. Heart Tregs in I/R injury models also have a prohealing phenotype similar to that of heart Tregs in MI models, probably because of the similar injured microenvironment in myocardium.

Previous studies indicate that CD4⁺ T cells first become primed and proliferate in the heart-draining MLNs after MI, and then infiltrate into injured hearts. These findings raise an important question: Where do heart Tregs take on their distinctive phenotypes, MLNs, or hearts? To address it, we compared transcriptomic characteristics between heart Tregs and MLN Tregs. The transcriptomic data showed that, differently from MLN Tregs, heart Tregs displayed a distinct repertoire signature dominated by a prohealing phenotype, which was attributable to the myocardial milieu under the scenario of myocardial injury. A recently published study also consistently pinpoints that T cells infiltrating the infarcted myocardium display a unique repertoire signature and clonally expand in situ.³² However, we also noticed that genes encoding Treg suppressive molecular Ctla4 and some cytokines, cytokine receptors, chemokines, and chemokine receptors were upregulated in MLN Tregs in comparison with spleen and NDLN Tregs. Therefore, we conclude that heart Tregs comply with a pragmatic lymphocyte-trafficking paradigm in a way that Tregs become primed and proliferative in MLNs. More importantly, the injured myocardium imposes an activation context that promotes the development of heart Tregs poising to exhibit a cardioprotective phenotype during the healing stage. It is notable that a recent study provides valid evidence to demonstrate that tissue Tregs are initially priming in lymph nodes and give rise to fully mature tissue Tregs in nonlymphoid tissues, exactly supporting our conclusion.³³

Heart Tregs Are Mainly Recruited From Circulation, Coupled With Proliferation and Conversion, and the IL-33/ST2 Axis Promotes Their Expansion

Tissue Tregs may have several different sources, including recruitment from peripheral Treg pool, amplification of recruited or locally colonized Tregs, and conversion from Tconvs.²⁵ Although some researchers have speculated that Tregs are mainly recruited from the peripheral circulation after MI,^12,13,34 relevant evidence is missing. Through FTY720 and photoconversion experiments, we confirmed the migration of circulating Tregs to the hearts after MI. More importantly, we calculated that 95% of the increased Treg population in the MI myocardium came from the peripheral circulation through parabiosis experiments. In comparison with lymphoid organs, the preferential aggregation of Tregs in the injured hearts may depend on their upregulated expression of chemokines and chemokine receptors as suggested by transcriptomic data.

In addition, it cannot be ignored that the other 2 processes also contributed to supplementing the Treg pool. Proliferative markers confirmed that heart Tregs exhibited heightened proliferation, which is consistent with visceral adipose tissue Tregs and muscle Tregs.^5,6,16,26 In particular, TCR repertoire data revealed that the clonal expansions of heart Tregs occurred in response to MI and were more pronounced in heart Tregs than in spleen Tregs, which suggests the possibility of specific antigen-TCR recognition by heart Tregs. Although heart Tregs expressed high levels of markers of thymus-derived Tregs, our results suggested that a minor proportion of heart Tregs was converted from Tconvs. By using the heart-specific antigen transgenic mice, Rieckmann et al³² also consistently found that Tconvs can be transformed into Tregs in MI. Otherwise, there was no conversion of visceral adipose tissue Tregs from Tconvs, indicating the distinct sources of individual tissue Tregs.²⁶

Given the strongly upregulated expression of ST2 on heart Tregs, we speculated that IL-33 drove heart Tregs enrichment. As expected, we verified that the IL-33/ST2 axis played an important role in the accumulation of heart Tregs through both gain- and lose-of-function experiments, which was consistent with our recent report on abdominal aortic aneurysm.³⁵ Regarding the mechanism of IL-33–induced Treg expansion, we showed that IL-33 injection enhanced the proliferation of Tregs. Another possible mechanism is whether IL-33 promoted heart Treg recruitment from circulation. In injured skeletal muscle, Kuswanto et al¹⁶ ruled out this possibility. Because most heart Tregs (≈90%) were in cell cycle, we considered that amplification rather than recruitment was the major contributor to IL-33/ST2 action on heart Tregs. Previous studies have demonstrated that IL-33 attenuates cardiac remodeling after MI,^36,37 which is consistent with a protective role of Tregs. Here, we showed that IL-33 significantly expanded heart Tregs, which represented a possible mechanism underlying the protection of IL-33 on MI.

Heart Tregs Promote Scar Repair, With Sparc as a Functional Mediator

The transcriptomic data showed an upregulation of extracellular matrix proteins and collagen-related genes of heart Tregs, indicating enhanced ability to promote the repair after MI. Thus, considering the traits of tissue Tregs, heart Tregs are more likely to play a cardioprotective role by directly promoting repair in relation to suppressing inflammation. Among these differentially expressed transcripts, Sparc is a prime example that has little expression in the lymphoid Tregs but is highly expressed by heart Tregs. Its corresponding product Sparc is an important cellular matrix protein that mediates cell-matrix interactions and regulates the production and assembly of extracellular matrices.³⁸ In mice, the absence of Sparc increases the rate of mortality attributable to heart rupture and dysfunction after MI.²⁸ This effect is mainly caused by impaired granulation tissue formation and collagen maturation after Sparc knockout.²⁸ Our data demonstrated that Sparc overexpression decreased mortality and cardiac rupture in Treg-ablated mice, which was associated with increased collagen content and enhanced maturation in the infarct scars, making up for the defects in tissue repair caused by Treg removal. However, except for Tregs, other Sparc-producing cells (including fibroblasts and macrophages) may also contribute to the effect of Sparc in MI settings. Thus, we further overexpressed Sparc on Tregs in vitro, and their effect on collagen was consistent with that in vivo. To directly address that Treg-derived Sparc is responsible for driving collagen synthesis and maturation in vivo, mice that specifically knock out Sparc in Tregs would be needed. In addition to Sparc, heart Tregs also highly expressed Areg, a functional mediator of Tregs in skeletal muscle⁶ and lung tissue,³⁹ and a group of extracellular matrix–related molecules, as well, which indicates that heart Tregs may function through multiple mediators. Whether these molecules also functionally contribute to heart Tregs remains to be determined.

Conclusions and Perspectives

In conclusion, we identified a unique population of heart Tregs with a prorepair phenotype that is mainly migrated from the circulation, coupled with local expansion and phenotypic conversion from Tconvs. Their accumulation is dependent on the IL-33/ST2 axis. These heart Tregs show strong expression of Sparc and play a necessary protective role in preserving cardiac integrity after MI by increasing collagen content and promoting maturation in the infarct scars. Harnessing the power of heart Tregs may therefore lead to the development of new treatment strategies for MI and other cardiac diseases.

Acknowledgments

We thank Prof McKenzie (Medical Research Council Laboratory of Molecular Biology, University of Cambridge, Cambridge, United Kingdom) for providing the Il1rl1^−/− mice. We also thank J. Min (Wuhan Institute of Virology, Chinese Academy of Sciences) for kindly assisting us with fluorescence-activated cell sorting.

Supplemental Materials

Data Supplement Methods

Data Supplement Tables I–III

Data Supplement Figures I–VIII

References 40–59

Footnotes