Dysfunctional and Proinflammatory Regulatory T-Lymphocytes Are Essential for Adverse Cardiac Remodeling in Ischemic Cardiomyopathy

Abstract

Background: Heart failure (HF) is a state of inappropriately sustained inflammation, suggesting the loss of normal immunosuppressive mechanisms. Regulatory T-lymphocytes (Tregs) are considered key suppressors of immune responses; however, their role in HF is unknown. We hypothesized that Tregs are dysfunctional in ischemic cardiomyopathy and HF, and they promote immune activation and left ventricular (LV) remodeling.

Methods: Adult male wild-type C57BL/6 mice, Foxp3-diphtheria toxin receptor transgenic mice, and tumor necrosis factor (TNF) α receptor-1 (TNFR1)^-/- mice underwent nonreperfused myocardial infarction to induce HF or sham operation. LV remodeling was assessed by echocardiography as well as histological and molecular phenotyping. Alterations in Treg profile and function were examined by flow cytometry, immunostaining, and in vitro cell assays.

Results: Compared with wild-type sham mice, CD4⁺Foxp3⁺ Tregs in wild-type HF mice robustly expanded in the heart, circulation, spleen, and lymph nodes in a phasic manner after myocardial infarction, beyond the early phase of wound healing, and exhibited proinflammatory T helper 1-type features with interferon-γ, TNFα, and TNFR1 expression, loss of immunomodulatory capacity, heightened proliferation, and potentiated antiangiogenic and profibrotic properties. Selective Treg ablation in Foxp3-diphtheria toxin receptor mice with ischemic cardiomyopathy reversed LV remodeling and dysfunction, alleviating hypertrophy and fibrosis, while suppressing circulating CD4⁺ T cells and systemic inflammation and enhancing tissue neovascularization. Tregs reconstituted after ablation exhibited restoration of immunosuppressive capacity and normalized TNFR1 expression. Treg dysfunction was also tightly coupled to Treg-endothelial cell contact- and TNFR1-dependent inhibition of angiogenesis and the mobilization and tissue infiltration of CD34⁺Flk1⁺ circulating angiogenic cells in a C-C chemokine ligand 5/C-C chemokine receptor 5-dependent manner. Anti-CD25-mediated Treg depletion in wild-type mice imparted similar benefits on LV remodeling, circulating angiogenic cells, and tissue neovascularization.

Conclusions: Proinflammatory and antiangiogenic Tregs play an essential pathogenetic role in chronic ischemic HF to promote immune activation and pathological LV remodeling. The restoration of normal Treg function may be a viable approach to therapeutic immunomodulation in this disease.

Keywords: angiogenic cells; heart failure; inflammation; neovascularization; regulatory T cells.

Figure 1.

Representative flow cytometry scatter plots and group quantitation for CD3⁺CD4⁺Foxp3⁺ Tregs in the heart (A) and blood (B) from sham and HF mice at the time points indicated. Naïve non-surgical control (NS Ctrl) also shown. (C) Representative low and high magnification confocal images of CD4⁺ (red) and Foxp3⁺ (green) immunostaining in sham and HF hearts (border, remote and scar zones 8 w post-MI) and quantitation of CD4⁺Foxp3⁺ cells. DAPI (blue) was used to label nuclei. (D) Representative flow cytometry scatter plots and quantitation for Tregs in the spleen and lymph nodes (LNs) from mice 8 w after MI or sham operation. (E) Representative flow plots for BrdU in cardiac Tregs (Left) and quantitation of total BrdU⁺ Tregs in the heart, blood, spleen and LNs 8 w after MI or sham operation. (F) LV (remote zone) and splenic gene expression of IL-2 and IL-6, and IL-2 and IL-6 serum levels, in sham and HF mice (8 w post-MI). Statistical comparisons: 2-tailed unpaired t-test at each time point except for A and B (right panel), 2-way ANOVA and Bonferroni post-test performed after (A) or without (B) logarithmic data transformation. N = 4–10/group. *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. sham.

Figure 2.

(A) Representative peripheral blood scatter plots and quantitative data for CD3⁺CD4⁺Foxp3⁺TNF⁺ Tregs in the blood, heart and spleen of sham and HF mice (heart and spleen measured 8 w post-operation); n = 4–7/group. (B) Single cell images for HF splenic Tregs expressing Foxp3 (pink) and CD4 (red), exhibiting variable expression of TNFR1 (green) and TNF (yellow). Also shown are DAPI nuclear staining (blue) and brightfield images. (C) Scatter plots and quantitation of cardiac TNFR1⁺ Tregs at various time points post-operation (Top). Frequency of circulating TNFR1⁺ Tregs in sham and HF mice at the time points indicated, and in the spleen and LNs 8 w post-operation (Bottom); n = 4–8/group. (D) In vitro Treg immunosuppression assay using splenic Tregs and Teffs isolated from naïve, sham and HF Foxp3-DTR (untreated) mice. Each experiment was conducted 3–4 times in triplicate using cells isolated from 3–4 mice. Statistical comparisons: 2-tailed unpaired t-test at each time point except for C (upper right panel) and D, 2-way ANOVA and Bonferroni post-test performed after (C) or without (D) logarithmic data transformation. *p < 0.05, **p <0.01, ***p<0.001, ****p<0.0001 vs. sham. ^#p < 0.05, ^###p<0.001 vs. naive.

Figure 3.

Representative whole hearts and transverse LV trichrome-stained sections (A) and gravimetric data normalized to tibia length (B) from sham and HF Foxp3-DTR mice treated with either DT or PBS vehicle as outlined in Figure S8A. N = 7–14/group. (C) Representative parasternal long-axis 2D echocardiograms at end-diastole and end-systole from Foxp3-DTR mice 8 w post-MI (HF) or sham operation after treatment with DT or PBS. (D) Group data for end-diastolic and end-systolic volume (EDV and ESV) and ejection fraction (EF) from Foxp3-DTR HF mice 4 and 8 w post-MI during 4 w of treatment with either DT or PBS. Statistical comparisons: B, 2-way ANOVA (Bonferroni post-test); D, 2-tailed paired t-test. N = 13–14/group. *p < 0.05, **p<0.01, ***p<0.001.

Figure 4.

(A) Representative Masson’s trichrome stains and group quantitation of peri-infarct interstitial fibrosis (% area) in Foxp3-DTR sham and HF hearts treated with either DT or PBS as in Figure 3. (B) LV (remote zone) collagen-I and collagen-III gene expression in the same groups. Statistical comparisons: 2-way ANOVA (Bonferroni post-test). N = 5–6/group; *p < 0.05, **p<0.01, ***p<0.001.

Figure 5.

(A) Representative cardiac isolectin (green) and WGA (red) staining, and quantitation of capillary:myocyte ratio and myocyte cross-sectional area in remote zone LV from sham and HF Foxp3-DTR mice given either DT or PBS vehicle. (B) Representative flow scatter plots for CD34⁺Flk1⁺ CACs and quantitation of circulating levels at 1 and 4 w after initiating DT or PBS as per the protocol in Figure S8A (i.e., 5 and 8 w post-MI or sham operation). (C) Frequency of CD34⁺Flk1⁺ CACs in the heart, spleen, LN and bone marrow (BM) in the same groups at 8 w. (D) Absolute CAC counts in blood (5 and 8 w post-MI), and heart, spleen, LN, and BM of Foxp3-DTR HF mice given DT or PBS at 8 w post-MI. (E) LV gene expression (remote zone) of CCR5 and CCL5 in sham and HF Foxp3-DTR mice treated with either DT or PBS. Statistical comparisons: A-C and E, 2-way ANOVA (Bonferroni post-test); D, 2-tailed unpaired t-test. N = 5–9/group; *p < 0.05, **p<0.01, ***p<0.001.

Figure 6.

(A) Frequency of circulating Tregs in sham and HF Foxp3-DTR mice treated with either DT or PBS at 8 w post-MI or sham operation; n = 4–7/group. Frequency of TNFR1⁺ and TNFR2⁺ Tregs (B), and CD4⁺ and CD8⁺ T-cells (C), in HF Foxp3-DTR mice treated with DT or PBS at the same time point; n = 4–7/group. (D) In vitro Treg immunosuppression assay using splenic Tregs and Teffs isolated from untreated HF Foxp3-DTR mice, HF Foxp3-DTR mice treated with DT, and untreated TNFR1^−/− HF mice. All cells were isolated 8 w post-MI, and the data for HF Foxp3-DTR mice are the same as in Figure 2D. Each experiment was conducted 3–4 times in triplicate using cells isolated from 3–4 mice. Statistical comparisons: A and D, 2-way ANOVA (Tukey’s post-test); B and C, 2-tailed unpaired t-test. *p < 0.05, **p <0.01, ***p<0.001 vs HF or as depicted; ^##p <0.01, ^###p<0.001 vs HF TNFR1^-/−.

Figure 7.

(A) Quantitative group data at the post-MI time points indicated for LV EDV, ESV, and EF in C57BL/6 HF mice treated with either anti-CD25 or isotype IgG control as per the protocol in Figure S14A. Representative cardiac isolectin (green) and WGA (red) staining, and quantitation of capillary:myocyte ratio (B) and LV remote zone gene expression of VEGF-A and CCR5 (C) in hearts from sham and HF mice (8 w post-MI) treated with either isotype IgG or anti-CD25 antibody. (D) Frequency and absolute counts of CD34⁺Flk1⁺ CACs in blood (5 and 8 w post-MI), and in the heart, spleen, BM, and LN at 8 w post-MI in anti-CD25- or IgG-treated HF mice. Statistical comparisons: A, 2-tailed paired t-test; B and C, 1-way ANOVA (Tukey’s post-test); D, 2-tailed unpaired t-test at each time point. N = 4–8/group; *p < 0.05, **p <0.01 vs sham or as depicted.

Figure 8.

(A) Representative brightfield images of tube formation by MCECs cultured alone and in the presence of CD4⁺CD25⁺eGFP⁺(Foxp3⁺) Tregs and CD4⁺CD25^–eGFP^–(Foxp3^–) Teffs isolated from Foxp3-DTR HF mice 8 w post-MI, or CD4⁺CD25⁺ Tregs and CD4⁺CD25^– Teffs from TNFR1^−/− HF mice. (B) Quantitation of total MCEC tube length under the various conditions indicated as detailed in the methods. Each experiment was conducted 3–4 times in triplicate using cells isolated from 3–4 mice. Statistical comparisons were performed using 1-way ANOVA (Tukey’s post-test). *p < 0.05, **p <0.01, ***p<0.001.