Exercise Training Protects Against Heart Failure Via Expansion of Myeloid-Derived Suppressor Cells Through Regulating IL-10/STAT3/S100A9 Pathway
Abstract
Background:
Exercise training (ET) has a protective effect on the progression of heart failure, however, the specific mechanism has not been fully explored. Myeloid-derived suppressor cells (MDSCs) are a group of myeloid-derived immunosuppressive cells, which showed a protective effect in the progression of heart failure. Thus, we hypothesized that the protective effect of ET on heart failure may be related to the infiltration of MDSCs.
Methods:
C57BL/6 mice were made to run on a treadmill 6× a week for 4 weeks followed by isoproterenol injection from third week. Heart function was evaluated by echocardiography and the proportion of MDSCs was detected by flow cytometry. Hypertrophic markers, cardiac fibrosis, and inflammatory factors were detected by real-time PCR, ELISA, histological staining, and Western blot.
Results:
ET treatment in isoproterenol-induced heart failure mice (n=7) enhanced cardiac function (57% increase in FS%, P=0.002) and improved morphological changes compared with isoproterenol mice (n=17). ET further caused 79% increasing in cardiac MDSCs in isoproterenol mice (P<0.001). In addition, depletion of MDSCs by 5-Fluorouracil blunted the cardio-protective effect of ET. T-cell proliferation assay showed that ET did not affect the suppressive activity of MDSCs. Furthermore, we found that ET activated the secretion of IL (interleukin)-10 by macrophages in isoproterenol mice. MDSCs expansion and cardio protection was not present in tamoxifen-inducible macrophage-specific IL-10 knockout mice. Western blot results confirmed that IL-10 regulated the differentiation of MDSCs through the translocation of p-STAT3 (signal transducer and activator of transcription 3)/S100A9 (S100 calcium-binding protein A9) to the nucleus.
Conclusions:
ET could increase MDSCs by stimulating the secretion of IL-10 from macrophage, which was through IL-10/STAT3/S100A9 signaling pathway, thereby achieving the role of heart protection.
What Is New?
1.
Exercise training could increase the proportion of myeloid-derived suppressor cells (MDSCs), which is linked to the cardio-prevention in heart failure mice.
2.
Macrophage-derived IL (interleukin)-10 contributes to exercise training induced MDSC expansion.
3.
IL-10 regulates the differentiation of MDSCs through translocation of p-STAT3 (signal transducer and activator of transcription 3)/S100A9 (S100 calcium-binding protein A9) to nucleus.
What are the Clinical Implications?
1.
The increase of MDSCs represents a new mechanism of myocardial protection induced by exercise training and may become an index to evaluate the effect of exercise training.
2.
Stimulating IL-10 secretion and then inducing the increase of MDSCs may be a promising therapeutic strategy for the treatment of heart failure.
Heart failure (HF) is a common cardiovascular disease with high morbidity and mortality.1 HF is a clinical syndrome caused by structural and functional defects in myocardium resulting in impairment of ventricular filling or the ejection of blood,2 usually caused by ischemic disease, but may also be caused by hypertension or valvular heart disease. The course of which is progressive damage, even if there is no myocardial damage, the degree of HF will gradually increase.
Recent studies have shown that immune activation and excessive production of inflammatory cytokines play an important role in the development of HF.3 At present, there are various methods to successfully reduce the damage of cardiomyocytes and prevent the development of HF by inhibiting the inflammatory response. These methods include blocking proinflammatory factors and chemokines,4 enhancing phagocytosis of dead cells, inhibiting changes in inflammatory receptors, and inhibiting myeloid cell recruitment and expansion.5,6 However, at present, a variety of treatments that directly improve the inflammatory state of patients with HF have not yet achieved satisfactory clinical results.
A large number of clinical and experimental data have confirmed that physical exercise can reduce the occurrence and development of chronic HF through its natural and powerful inflammatory inhibition, but its molecular mechanism for inhibiting inflammatory response is still inconclusive.7–9 Existing evidence shows that exercise can effectively reduce the levels of inflammatory factors IL (interleukin)-1β, TNF-α (tumor necrosis factor-α), CRP (C-reaction protein) in the serum of patients with coronary heart disease, and effectively increase the content of anti-inflammatory factors IL-10, IL-1ra, and other cytokines.10 Correspondingly, in the absence of exercise intervention, the treatment of HF with the IL-1 receptor antagonist-anakinra can effectively improve the exercise tolerance of patients.11 Interestingly, exercise can significantly reduce the expression of skeletal muscle inflammatory cytokines, so its protective effect on HF may be related to cytokines in blood circulation.12 However, the molecular mechanism of exercise training (ET) to suppress the inflammatory response during HF is inconclusive. Therefore, it is particularly important to further explore the protective mechanism of exercise in patients with HF.
Myeloid-derived suppressor cells (MDSCs) are a highly heterogeneous population of immature myeloid cells with immunosuppressive function.13,14 MDSCs can be characterized by the expression of CD11b+Gr-1+ in mouse. In healthy individuals, MDSCs are generated in bone marrow and quickly differentiate into mature dendritic cells, macrophages, or granulocytes. However, under pathological conditions, the differentiation of MDSCs will be blocked and cause the expansion of this population in vivo.15 Recent studies have shown that in the case of HF, MDSC will spread to the bone marrow, blood, and heart. But the increase of MDSC has a protective effect on the heart.16 Therefore, the functional research of MDSC needs to be further explored. Whether exercise is related to the increase of MDSC while protecting the heart is still unknown.
In this study, we found that ET could effectively inhibit HF induced by isoproterenol treatment. In addition, ET could further increase the proportion of MDSCs in HF mice. Depletion of MDSCs blunted the protective effect of ET on HF. Further study confirmed that IL-10/STAT3 (signal transducer and activator of transcription 3)/S100A9 (S100 calcium-binding protein A9) mediated the effects of ET on MDSCs expansion. Deletion of IL-10 in macrophages reversed MDSCs expansion and cardiac protection induced by ET. Our findings suggested that MDSC can be used as a new treatment strategy in the discovery and treatment of HF.
Methods
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Animal Models
Male C57BL/6 mice (6–8 weeks old) weighing 18 to 22 g were purchased from SPF(Sibeifu) Biotechnology (Beijing, China). Room temperature was maintained at 22 °C–24 °C, and mice were housed on a 12-hour light-dark cycle. All animal care and experimental Protocols were approved by Institute Research Ethics Committee of Nankai University (Permit number: 10011) and followed strictly the guidelines for laboratory animals in Nankai University. For HF disease models, isoproterenol (30 mg/kg; Sigma-Aldrich, St. Louis, MO) in saline was infused with subcutaneous injection daily for 2 weeks. The ET protocol was based on a previous protocol.17
Briefly, mice were placed in treadmill fitted with running wheels (China, YLS-10B) for a period up to 4 weeks. Mice ran 6× a week, the sessions initially lasted for 1.5 hours and were increased by 15 minutes each day to reach 2.5 hours on day 5. The control and isoproterenol groups remained sedentary during the first 2 weeks. According to previous study,16 isoproterenol (30 mg/kg) was injected subcutaneously once daily for 14 days to induce HF. For ET+isoproterenol group, isoproterenol were injected subcutaneously at 8:00 am and exercised for 2.5 hours at 8:00 pm until the end of the experiment. To confirm mouse HF model, left ventricle (LV) systolic function such as ejection fraction (EF), fractional shortening, and cardiac output and LV hypertrophy such as and atrial natriuretic factor (ANF) expression, LV mass, and interstitial fibrosis were detected.
Mononuclear Cells Isolation
Peripheral blood mononuclear cells were isolated from the fresh heparinized peripheral blood of C57/BL6 mice used Mouse Peripheral Blood Mononuclear Cell Separation Solution KIT (Haoyang Bio, Tianjin) following the kit instructions.
Spleens were removed after the last isoproterenol subcutaneous injection. The cells were dispersed with a syringe plunger. The cell suspension was then filtered through 40 μm cell strainers, and erythrocytes were lysed with 0.83% ammonium chloride lysis solution. Splenocytes were washed and resuspended.
We use a syringe to flush out bone marrow cells from mouse femurs and tibias with PBS, and lyse the erythrocytes with lysis solution, and resuspend to make bone marrow cell suspension.
For the separation of cardiac single cells, we used the Langendorff device to digest the heart with type II collagenase, tore the heart apart with tweezers, and gently beat it evenly with a straw. After natural precipitation for 15 minutes, cardiac cells were precipitated and the rest were single-cell suspension. The single-cell suspension was taken and passed through a 40 μm mesh sieve. After centrifugation, the monocytes were isolated using the Mouse Organ Tissue Mononuclear Cell Separation Solution KIT (Haoyang Bio, Tianjin) according to the instructions.
Quantitative Real-Time Polymearse Chain Reaction
The protocol for quantitative real-time polymearse chain reaction has been described previously.18 In brief, total RNA was isolated using Trizol reagent (Takara Bio, Japan) from mouse heart tissues. mRNA (1.0 μg) was used for cDNA synthesis by reverse transcription system (TransGen Biotech, China). Real-time polymearse chain reaction was performed using SYBR Green Master Mix (TransGen Biotech, China) in a Roche LightCycler 96 detection system. The cycle threshold values were automatically determined in triplicates and averaged.
Western Blot Analysis
Left ventricle in tissue and cells sample were lysed in ice-cold RIPA lysis buffer and centrifuged at 12 000g at 4 °C for 30 minutes. Total protein concentrations were measured by BCA protein assay Kit (Thermo, United States).The protein sample (20–50 µg) was separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membrane (Merck KGaA, Darmstadt, Germany). After blocking with 5% BSA for 2 hours, membranes were incubated overnight at 4 °C with primary antibodies, including anti-IL-10 (abcam), anti-p-STAT3 (CST), T-STAT3 (CST), anti-S100A8 (Thermo Fisher Scientific), anti-S100A9 (Thermo Fisher Scientific), and anti-β-Tubulin (santa cruz) followed by horseradish peroxidase-conjugated secondary antibodies. The results were expressed as fold changes normalized to β-Tubulin.
MDSC Isolation
Murine MDSCs were separated from spleens 2 weeks after isoproterenol infusion. Briefly, mouse splenocytes were fractionated by Mouse Organ Tissue Mononuclear Cell Separation Solution KIT (Haoyang Bio, Tianjin). CD11b+ GR1+ MDSCs were isolated with Mouse MDSC Isolation Kit according to the manufacturer’s instructions. The purity of MDSC is >90% by flow analysis.
Isolation of Neonatal Rat Primary Cardiomyocytes
Hearts from neonatal rats (1–3 days old) were cut into small pieces in ice-cold Hanks’ balanced salt solution and then digested overnight with trypsin at 4 °C. Next day, the heart pieces were digested with type II collagenase at 37 °C.Cardiomyocytes were collected after centrifugation at 500g for 5minutes, followed by removal of fibroblasts and endothelial cells through adherence to tissue culture dishes for 75 minutes at 37 °C twice.
Coculture of MDSCs and Cardiomyocytes
Neonatal Rat Primary Cardiomyocytes were routinely cultured in DMEM with 1% ITS at 37 °C in a 5% CO2 atmosphere. MDSCs were cocultured with primary cardiomyocytes at a ratio of 5:1.
For Transwell experiments, 2×105 primary cardiomyocytes were seeded in each upper chamber of a Transwell system (Corning, Lowell, MA) with a polycarbonate membrane (0.4 μm pore size), and 106 MDSCs were seeded in each lower chamber. Isoproterenol (10 µmol/L) was added to the upper chambers, followed by incubation for 24 hours.
Immunofluorescence Microscopy
Heart and spleen tissue sections or primary cardiomyocytes seeded on coverslips were permeabilized with 0.5% Triton X-100 for 15 minutes at RT, blocked with 5% goat serum for 1 hour at RT, and incubated with primary antibodies (α-actin [Thermo Fisher Scientific]), cd11b (abcam), GR-1 (R&D) overnight at 4 °C. Samples were then washed in TBST and incubated with appropriate Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (1:200, CST) for 1 hour at RT. Nuclei were stained with DAPI (Sigma) for 2 minutes at RT. Images were acquired with a FV1000 confocal microscope (Olympus) and analyzed with ImageJ.
Statistical Analysis
All the data were presented in box-plot. Statistical analysis was performed by using Graphpad Prism 9. Data were tested for normality using the Shapiro-Wilk test. Data that were normally distributed were analyzed with 1-way or 2-way ANOVA followed by multiple comparisons performed with LSD test among 3 or more groups, followed by unpaired Student t tests with the Bonferroni correction between 2 groups. The Kruskal-Wallis 1-way ANOVA on ranks was used for data that were not normally distributed. A value of P<0.05 was accepted as statistically significant.
Results
ET Inhibited HF Induced by Chronic Isoproterenol Treatment
To detect the effect of ET on morphological changes induced by isoproterenol injection, echocardiographic analysis was performed after the last isoproterenol injection. As shown in Figure 1, mice with isoproterenol injection showed marked impairment in cardiac contractile function as represented by decrease in left ventricular fractional shortening, EF, cardiac output, and stroke volume (Figure S1). Conversely, compared with isoproterenol group, there was apparent increased of FS (57% increased, P=0.002), EF (32% increased, P=0.003), cardiac output (57% increased, P=0.026), and stroke volume (61% increased, P<0.001) in ET+isoproterenol group. In addition, isoproterenol treatment resulted in significant increase in heart weight/body weight (HW/BW) ratio, HW/tibia length, ANF expression, and LV mass. HW/BW (17% decreased, P<0.001), HW/tibia length (22% decreased, P<0.001), ANF expression (76% decreased, P<0.001), and LV mass (21% decreased, P=0.002) were also decreased obviously in ET+isoproterenol group compared with isoproterenol group (Figure 1F through 1H, Figure S1C). Heart rate in isoproterenol group was lower than that in control group, and there was apparent increase in heart rate in ET+isoproterenol group compared with isoproterenol group (Figure S1D, P=0.001). Moreover, HE staining displayed a significant increase in cross-section area of ventricular myocardium in isoproterenol-treated mice, whereas the cross-section size was significantly decreased by ET treatment (P<0.001). Masson staining and Sirius red staining showed that interstitial fibrosis was markedly increased in isoproterenol-treated mice, and these alterations were prevented by ET (Figure 1I through 1L, P=0.013 and P<0.001, respectively). These data indicate that ET could inhibit isoproterenol-induced myocardial hypertrophy in mice.

Figure 1. The effect of exercise training (ET) on isoproterenol (ISO)-induced pressure-overloaded heart failure.
A, Experimental outline. Mice were housed in cages fitted with running wheels and allowed to ET for 4 weeks. From the third week, mice were subcutaneously injected with ISO for 14 days (ET+ISO, n=7). Isoproterenol mice (ISO, n=17) or control mice (CON, n=13) were housed in cages without running for the same durations as were the ET mice and then injected with ISO or vehicle control. ET mice (n=9) were allowed to ET for 4 weeks. B, Representative echocardiographic image for each group. C through E, Quantitative analysis of ejection fraction, fractional shortening, and cardio output for each group. F and G, Heart weight/tibia length (HW/TL) and heart weight/body weight (HW/BW) were calculated. H, Relative quantitative analysis of atrial natriuretic factor (ANF) mRNA levels in heart tissue. I, Representative hematoxylin and eosin (HE)–stained (scale bars, 50 μm), Masson-stained (scale bars, 100 μm), and Picrosirius Red–stained (scale bars, 100 μm) heart sections. J through L, Relative quantitative analysis of cardiomyocyte area, fiber area, and Picrosirius Red area for each group. All of the data were presented in box plots. All box plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. UCG indicates ultrasound cardiography.
ET Caused MDSCs Elevation in HF Mice
To explore the mechanism of ET on modulating HF progression, murine MDSCs (CD11b+GR1+) were detected by flow cytometry. It was found that the percentages of MDSCs were significantly increased in bone marrow, peripheral blood, spleen and heart in isoproterenol group (P=0.015, P=0.030, P=0.002, P=0.005, respectively, versus control, Figure 2A through 2D). Interestingly, the proportion of MDSCs in ET+isoproterenol group was observed more frequently than that of isoproterenol group in immune organs and heart (79% increased, P<0.001, Figure 2A through 2D). In addition, immunofluorescence staining results showed that infiltration of CD11b+ (green) and GR1+ (red) co-expressing MDSCs (yellow) was further increased in spleen in ET+isoproterenol group compared with isoproterenol group (47% increased, P=0.006, Figure 2E). Noticeably, in the absence of isoproterenol, the percentage of CD11b+ GR1+ cells in ET group were not increased significantly, which may be explained by the adaption to ET. These results indicated that ET could cause murine MDSCs elevation in isoproterenol-treated mice. Furthermore, as mature myeloid cells, the proportion of macrophages in heart and peripheral blood mononuclear cells were also detected in HF mice. As shown in Figure S1E and S2, the percentage of macrophages was systematically upregulated in HF mice and ET could significantly reverse this change in ET+isoproterenol group. These results suggested that ET may regulate the differentiation of myeloid cells.

Figure 2. Exercise training (ET) caused myeloid-derived suppressor cells (MDSCs) expansion in the peripheral blood mononuclear cell (PBMC), bone marrow (BM), spleen, and heart in isoproterenol (ISO)-treated mice.
A through D, Flow cytometry analysis of MDSCs (CD11b+GR1+) in PBMC, BM, spleen, and heart (n=6–8 mice per group). E, Immunofluorescence staining for CD11b (red) and GR1 (green) in spleen. CD11b+GR1+ MDSCs infiltrated in spleen were shown in yellow (×800 magnification). Images are representative of 3 independent experiments. All the data were presented in box plots. All box plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. CON indicates control; DAPI, 4′,6-diamidino-2-phenylindole; and ISO, isoproterenol.
MDSCs Mediated the Protective Effect of ET on Cardiac Protection
To address whether the cardio protective effect of ET was related to MDSCs, 5- fluorouracil (5FU; 50 mg/kg) was used to deplete endogenous MDSCs before ET according to previous study.16 As shown in Figure 3B, MDSCs significantly decreased in peripheral blood by 5FU treatment in ET+isoproterenol+5FU group compared with ET+isoproterenol group. In accordingly, MDSCs depletion by 5FU significantly blunted the beneficial effects of ET on isoproterenol-induced cardiac hypertrophy, as manifested by increased HW/BW ratio (P<0.001), myocyte area (P<0.001), and myocardial fibrosis area (P<0.001, ET+isoproterenol versus ET+isoproterenol+5FU, Figure 3C, 3E through 3H). Moreover, the improvement in echocardiographic alterations in ET+isoproterenol group was also reversed by 5FU treatment, which was demonstrated by a significant decrease in EF (P=0.006, Figure 3C and 3I). These data demonstrated that MDSCs depletion could exacerbate the pathological progression of HF and MDSCs accumulation was associated with ET mediated cardio protection.

Figure 3. Depletion of myeloid-derived suppressor cells (MDSCs) by 5-fluorouracil (5FU) blunted the protective effect of exercise training (ET) on heart failure.
A, Experimental outline. Mice in ET+isoproterenol (ISO) group (n=4) were allowed to ET from week 1 to week 4 and were injected with ISO from week 3 to week 4. Mice in ISO group (n=6) or control (CON) group (n=5) were injected with ISO or vehicle at the same durations without ET. Three days after ISO injection, 5FU (50 mg/kg) was injected intraperitoneally once every 5 days (n=3–5 in each group). B, The analysis of MDSCs (CD11b+GR-1+) in peripheral blood mononuclear cell (PBMC) by flow cytometry. C, Representative hematoxylin and eosin (HE)–stained (scale bars, 50 μm), Masson-stained (scale bars, 100 μm), and Picrosirius Red–stained (scale bars, 100 μm) heart sections and echocardiographic images for each group. D, The proportion of MDSCs from flow cytometry. E, Heart weight/body weight (HW/BW) was calculated for each group. F through H, Relative quantitative analysis of cardiomyocyte area, fiber area, and Picrosirius Red area. I, Quantitative analysis of ejection fraction for each group. All the data were presented in box plots. All box plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. UCG indicates ultrasound cardiography.
ET Did Not Affect the Suppressive Activity of MDSCs
Next, to further verify the effect of ET on MDSCs’ activity, Splenic MDSCs in isoproterenol group and ET+isoproterenol group were isolated and cocultured with T cells. The results showed that both isoproterenol-MDSCs and ET+isoproterenol-MDSCs strongly inhibited the proliferation of autologous CD3+ T cells in the presence of anti-CD3/anti-CD28 antibodies (Figure 4A and 4B). However, there were no differences in the degree of inhibition of T cells by MDSCs isolated from these 2 groups.

Figure 4. Exercise training (ET) did not affect the suppressive activity of myeloid-derived suppressor cells (MDSCs).
A, Purified MDSCs were cocultured with splenocytes from isoproterenol (ISO)-treated mice at a ratio of 2:1 in the presence of anti-CD3/anti-CD28 dynabeads for 72 hours, and the proliferation of CD3+ T cells was analyzed by flow cytometry. B, The percentage of proliferated CD3+ T was shown as box plot from 3 independent experiments. C, A total of 106 purified MDSCs from ISO-treated mice were cocultured with 2×105 neonatal rat primary cardiomyocytes in the presence of ISO (10 μmol/L) for 24 hours at a ratio of 5:1 in direct-contact condition. Immunofluorescence staining for α-actin and atrial natriuretic factor (ANF) in cardiomyocytes (×600 magnification). D, Quantitative data of relative cell surface area analyzed by Image-Pro Plus 6.0 software. E, Lactate dehydrogenase (LDH) activity in the supernatant was detected. All the data were presented in box plots. All box plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum.
Previous studies reported that MDSCs isolated from HF mice could significantly suppress isoproterenol-induced hypertrophic response in myocardial cells in vitro.16 Thus, in addition to their immunosuppressive activity, the specific cardio protective function of MDSCs was also checked. Splenic MDSCs were isolated and cocultured with neonatal rat ventricular myocytes in transwell system and then stimulated by isoproterenol for 48 hours. It was found that MDSCs from both isoproterenol and ET+isoproterenol groups significantly suppressed the isoproterenol-induced hypertrophic response in neonatal rat ventricular myocytes, as shown by reduced cell size, ANF expression, and lactate dehydrogenase activity (Figure 4C through 4E). However, there were no differences between the isoproterenol-MDSCs and ET+isoproterenol-MDSCs groups. Taken together, the results indicated that ET did not affect the function of MDSCs.
IL-10/STAT3/S100A9 Mediated MDSCs Expansion Induced by ET
The proinflammatory cytokine IL-6 and immunosuppressive cytokine IL-10 play a major role in cardiac hypertrophy and were implicated in the generation and activation of Gr1+CD11b+ MDSCs under different inflammatory conditions.19,20 Thus, the expression of IL-6 and IL-10 in plasma and heart were measured in different time points (Figure 5A). As shown in Figure 5B and 5C, the levels of IL-6 increased rapidly in isoproterenol-treated mice on day 1 and last for 1 week after cardiac hypotrophy onset. However, in ET+isoproterenol group, the levels of IL-6 decreased significantly on day 7 compared with isoproterenol mice (P<0.05). Meanwhile, the IL-10 levels increased significantly in ET mice compared with isoproterenol mice on day 1. And the IL-10 expression still maintained high levels both in ET+isoproterenol and ET group after 7 days (P<0.05). These results indicated that the expansion of MDSCs in ET+isoproterenol mice was associated with elevated levels of IL-10 both in heart and serum.

Figure 5. IL (interleukin)-10/STAT3 (signal transducer and activator of transcription 3) mediated the effects of exercise training (ET) on myeloid-derived suppressor cells (MDSCs) expansion.
A, Experimental outline for B and C. Mice were allowed to ET and were injected with isoproterenol (ISO) for 1 day and 7 days. Blood and heart samples were collected at day 1 and day 7, respectively. B and C, The IL-6 and IL-10 levels in serum and heart tissues were determined by ELISA. D, Experimental outline for E through G. E, p-STAT3, total-STAT3, S100A8, S100A9, and IL-10 in myocardial tissues were analyzed by Western blot. F, The expression of p-STAT3, total-STAT3, and S100A9 in the cytoplasm and nucleus of purified MDSCs were detected by Western blot. G, The expression of p-STAT3 and S100A9 in the cytoplasm and nucleus of IL-10-stimulated naive MDSCs was analyzed by Western blot. All the data were presented in box plot. All box plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. CON indicates control; S100A8, S100 calcium-binding protein A8; and S100A9, S100 calcium-binding protein A9.
Next, the expression and phosphorylation of functional proteins were checked in heart tissue and Gr1+CD11b+ cells of HF mice (Figure 5D). Western blot results showed that the phosphorylation levels of STAT3 in the heart were obviously increased in isoproterenol and ET+isoproterenol mice compared with control mice (Figure 5E, P<0.001). In addition, the expressions of S100A8 (S100 calcium-binding protein A8) and S100A9, which have a crucial role in regulating MDSCs expansion, were also upregulated both in isoproterenol and ET+isoproterenol mice (P<0.001 versus control). However, there was no significant differences between these 2 groups. According to previous study, intracellular S100A9 was responsible for reprogramming Gr1+CD11b+ cells into MDSCs phenotype.21 Thus, the location of S100A9 was further determined by Western blot. Interestingly, the results revealed that both the phosphorylated STAT3 and S100A9 protein were mainly localized in the cytosol of Gr1+CD11b+ cells in isoproterenol group. However, the phosphorylated STAT3 and S100A9 protein were translocated to nuclear compartment of Gr1+CD11b+ cells in ET+isoproterenol group (Figure 5F). Then, to further confirm the critical role of IL-10 in promoting S100A9 nuclear localization, naive Gr1+CD11b+ was stimulated by IL-10 in vitro. As shown in Figure 5G, the phosphorylation level of STAT3 was increased significantly by IL-10 incubation and localized into the nuclear. Furthermore, in the absence of IL-10, S100A9 was mainly present in the cytosol, but localized into the nuclear with IL-10 stimulation. These results suggested that IL-10 induces nuclear localization of S100A9 in MDSCs through STAT3 pathway during HF.
Macrophage-Derived IL-10 Contributed to ET-Induced MDSC Expansion and Cardiac Protection
IL-10 is a pleiotropic anti-inflammatory cytokine produced and sensed by many cells, including macrophages.16,22–24 To trace the source of IL-10, heart cells in ET mice were isolated by langendorff perfusion. And after type II collagenase digestion, macrophages, endothelial cells, and fibroblasts were obtained by flow sorting. RT-PCR results showed that IL-10 were mainly expressed on macrophages compared with myocardial cell, cardiac fibroblasts, and endothelial cells, which indicated macrophages were indeed a dominant source of IL-10 in ET mice (Figure 6A and 6C). Then, to probe the contribution of macrophage-derived IL-10 to MDSCs accumulation and cardiac protection, we bred tamoxifen-inducible Cx3cr1CreER with IL10fl/fl mice, hereafter denoted as Cx3cr1 IL10−/− mice in which tamoxifen treatment could delete IL-10 in Cx3cr1-expressing monocytes and macrophages. After tamoxifen intraperitoneal injection daily for 7 days, genomic PCR-based examination of the wild-type (IL-10wt), floxed intact (IL-10fl), and recombined (IL-10Δ) alleles of the IL-10 gene in fluorescence-activated cell sorting-purified heart macrophages showed effective IL-10 deletion (Figure 6D and 6E). In addition, cardiac macrophages from Cx3cr1 IL10−/− mice and their IL-10 intact littermates (without tamoxifen injection) were isolated and the secretion and expression of IL-10 were detected by ELISA and RT-PCR. As shown in Figure 6F and 6G, IL-10 concentration in culture supernatants (P<0.001) and the mRNA level of IL-10 (P=0.001) in primary fluorescence-activated cell sorting-purified heart macrophages of Cx3cr1 IL10−/− mice were significantly decreased compared with littermate mice.

Figure 6. Validation of IL (interleukin)-10 produced by cardiac macrophages.
A, Experimental outline for cardiomyocyte purification. B, Macrophages, endothelial cells, and fibroblasts were separated by flow cytometry. C, The level of IL-10 in left ventricular cardiomyocytes, macrophages, fibroblasts, and endothelial cells were analyzed by real-time polymearse chain reaction (PCR). D, Experimental outline for conditional deleting IL-10 alleles. Cx3cr1 IL-10−/− mice were intraperitoneally injected with tamoxifen for 5 consecutive days, and then heart macrophages were collected and fluorescence-activated cell sorting (FACS)-purified 7 days after tamoxifen treatment. E, PCR analysis of FACS-purified Cx3cr1wt/wt and Cx3cr1wt/CreER heart macrophages 7 d after tamoxifen treatment for the presence of wild-type (IL-10wt) and conditional undeleted (IL-10fl) or deleted IL-10 alleles (IL-10Δ). F, IL-10 concentration in culture supernatants of primary FACS-purified heart macrophages. G, The mRNA level of IL-10 in primary FACS-purified heart macrophages. All the data were presented in box plots. All box plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum.
Next, to further explore the effect of macrophage-derived IL-10 on the cardioprotective effect of ET, Cx3cr1 IL-10−/− mice were exposed to isoproterenol injection and ET while receiving additional tamoxifen injection once a week to deplete IL-10 in newly made monocytes and monocyte-derived macrophages (Figure 7A). The IL-10 intact littermate mice were intraperitoneally injected with corn oil instead. Echocardiographic (Figure 7B and 7E) and histological (Figure 7C, 7D, 7F, and 7G) analysis showed that isoproterenol-induced marked impairment in EF, cardiac hypertrophy, and fibrosis were improved by ET in littermate mice (P<0.001, P=0.002, P<0.001, respectively). These ameliorations were not found in Cx3cr1 IL10−/− mice. As expected, there was a significant increase in IL-10 expression in ET+isoproterenol group compared with isoproterenol group in littermate mice (P=0.005) but not in Cx3cr1 IL10−/− mice exposed to isoproterenol and ET treatment (Figure 7H). HW/tibia length (Figure 7I) and ANF expression (Figure 7J) in heart tissue were significantly down-regulated in ET+isoproterenol group of littermate mice (P<0.001 and P=0.005, respectively) but not in Cx3cr1 IL10−/− mice. These data indicated that the protective effect of ET on HF was abolished in macrophage-specific IL-10 knockout mice.

Figure 7. Knockout of IL (interleukin)-10 in macrophages blocked the protective effect of exercise training (ET) in heart failure mice.
A, Experimental outline. Mice were intraperitoneally injected with tamoxifen for 5 days a week in advance. Littermate mice and Cx3cr1 IL10−/− mice were allowed to ET for 4 wk. From the third week, mice were subcutaneously injected with isoproterenol (ISO) for 14 days. Isoproterenol (ISO) mice were housed in cages without running for the same durations. (n=6–8 mice per group). B through D, Representative echocardiographic images, hematoxylin and eosin (HE)–stained (scale bars, 50 μm) and Masson-stained (scale bars, 100 μm) heart section. E, Quantitative analysis of ejection fraction for each group. F through G, Quantitative analysis of cardiomyocyte area and fiber area. H, The levels of IL-10 in serum were determined by ELISA. I, Heart weight/tibia length (HW/TL) was calculated. J, mRNA levels of atrial natriuretic factor (ANF) in heart tissue. K, MDSCs population in spleen, peripheral blood mononuclear cell (PBMC), and heart by flow cytometry analysis. L, The expression of p-STAT3, total-STAT3, and S100A9 in purified MDSCs by Western blot analysis. M, Purified MDSCs were cocultured with splenic cells from ISO-treated mice at a ratio of 2:1 in the presence of anti-CD3/anti-CD28 dynabeads for 72 h, and proliferation of CD3+ T cells was analyzed by flow cytometry. All box plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. MDSCs indicates myeloid-derived suppressor cells; S100A8, S100 calcium-binding protein A8; S100A9, S100 calcium-binding protein A9; STAT3, signal transducer and activator of transcription 3; and UCG, ultrasound cardiography.
Then, the regulated effect of ET on MDSCs were explored in Cx3cr1 IL-10−/− mice. In consistent with WT mice, ET-induced significant increase in the proportion of MDSCs in blood, spleen, and heart in littermate mice after isoproterenol treatment. However, MDSCs expansion was not found in Cx3cr1 IL10−/− mice. In addition, the translocations of p-STAT3 and S100A9 to the nucleus of MDSCs were attenuated in Cx3cr1 IL10−/− mice after isoproterenol treatment (Figure 7K and 7L). Finally, T-cell proliferation assay revealed that purified MDSCs isolated from Cx3cr1 IL10−/− mice could also suppress the proliferation of autologous CD3+ T cells (Figure 7M). These results implied that knockout of macrophage-derived IL10 blocked the protective effect of ET by attenuating the accumulation of MDSCs in HF mice.
The Therapeutic Effect of ET on HF Mice
Next, we further explored the therapeutic effect of ET on isoproterenol-induced HF mice as shown in Figure 8A. The results showed that ET improved EF (P=0.012) and reduced HW/BW in HF mice (P=0.008, versus isoproterenol mice; Figure 8B through 8D). Similarly, the expression level of ANF was also decreased by ET (Figure 8E; P<0.001, versus isoproterenol mice). The pathological sections of myocardial tissue showed that ET treatment also reduced the area of myocardial fiber (P=0.031, versus isoproterenol) and the degree of fibrosis caused by isoproterenol (P<0.001, versus isoproterenol; Figure 8F through 8H). In other hand, depletion of MDSCs could further aggravate cardiac dysfunction and hypertrophy as indicated by reduced EF and increased HW/BW, ANF expression, and interstitial fibrosis (Figure 8B through 8H). These results further proved that ET showed protective effect on HF and depletion of MDSC could aggravate the degree of HF.

Figure 8. Therapeutic effects of exercise training (ET) on isoproterenol (ISO)-induced heart failure.
A, Experimental outline. Mice were injected with ISO, 6–8 h later, followed by ET (ET+ISO, n=5). This scheme was repeated for 2 weeks. Mice in ISO group were just injected with ISO without ET (n=6). On day 3, 5-fluorouracil (5FU; 50 mg/kg) was injected intraperitoneally once every 5 days (ISO+5FU, n=5). B, Representative echocardiographic images. C, Quantitative analysis of ejection fraction. D, Heart weight/body weight (HW/BW) was calculated. E, Quantitative analysis of atrial natriuretic factor (ANF) mRNA levels. F, Representative hematoxylin and eosin (HE)–stained (scale bars, 50 μm) and Masson-stained (scale bars, 100 μm) heart section. Relative quantitative analysis of cardiomyocyte area (G) and fiber area (H) for each group. I, The overview diagram of the article to show how exercise training protects the heart through myeloid-derived suppressor cells (MDSCs). All the data were presented in box plots. All box plots show the minimum, the 25th percentile, the median, the 75th percentile, and the maximum. CON indicates control; IL, interleukin; TL, tibial length; and UCG, ultrasound cardiography.
Taken together, our results demonstrated that ET activated the secretion of IL-10 by macrophages in HF mice and promoted the translocation of p-STAT3/S100A9 to the nucleus to regulate the differentiation of MDSCs, thus achieving cardiac protection (Figure 8I).
Discussion
ET has been recommended as an important component of therapy for chronic HF patients.25 Nevertheless, the underlying molecular mechanisms responsible for the benefit of exercise are still far from clear. In this study, we demonstrated that ET further increased the proportion of MDSCs in pressure overload-induced HF mice. MDSCs depletion aggravated isoproterenol-induced cardiac hypertrophy, fibrosis, and dysfunction in ET mice. IL-10/STAT3/S100A9 mediated MDSCs expansion induced by ET. Knockout of IL-10 in macrophages reduced the percentage of MDSCs and reversed the protective effect of ET in HF mice.
In our previous research, we found that proper ET can inhibit the cardiac hypertrophy caused by isoproterenol.17 In this study, isoproterenol-induced HF model was used to test the effect of ET on HF. Isoproterenol model is a well-established HF model, and it is convenient to generate and use.16,26 In this model, animal manifest prominent cardiac hypertrophy, ventricular dysfunction and massive cell death with replacement fibrosis, which recapitulates the major aspects of human HF. In our study, we found consistent and reproducible morphological and echocardiographic alterations characterizing HF, which justify the suitability of this model for our purpose. In the future, it would be desirable to use additional HF models to test our results such as thoracic aortic constriction, which is widely accepted and of clinical relevance. In addition, this study mainly focuses on ET intervention when hearts are healthy. To further test how ET protects against HF progression, mice were injected with isoproterenol followed by ET 6 to 8 hours later. As shown in Figure 8, treatment with ET greatly reversed isoproterenol-induced significant decrease in EF, significant increase in HW/BW ratio and ANF expression. These results confirmed the therapeutic effect of ET on mice with HF. However, whether ET can increase the proportion of MDSCs in HF mice needs to be further confirmed.
Previous studies have shown that MDSC numbers were significantly elevated in patients with HF and in pressure overload-induced HF mouse models.16 In this study, we also found that the percentages of MDSCs were markedly increased in immune organs and heart. Interestingly, the proportion of MDSCs increased more significantly in ET+isoproterenol group. Based on the fact that exercise has a protective effect on the heart, it is not difficult to speculate that ET may have a protective effect on the heart by further increasing the number of MDSCs in mice with HF (Figure 2). In addition, 5FU was used to deplete endogenous MDSCs without significant effect on T cells, B cells, and dendritic cells according to previous study.27 Therefore, we used 5FU to deplete the MDSCs in mice and found that the proportion of MDSC in peripheral blood decreased significantly. At the same time, the protective effect of exercise on the heart was significantly weakened. In addition, 5FU treatment aggravated the degree of HF in isoproterenol group (Figure 8). All the results indicated that MDSCs expansions contributed to ET-induced cardio-prevention in HF mice.
MDSCs was an immunosuppressive cell, its main role is to suppress the immune response, a large number of studies have confirmed its inhibitory effect on T cells.28–30 T-cell immune response leads to acute and chronic inflammatory processes that damage heart function through direct cytotoxicity or by enhancing the inflammatory function of other cells. This can lead to permanent damage to the heart tissue, which is then replaced by fibrosis, and often leads to dilated cardiomyopathy with congestive HF.31 Therefore, the inhibition of over-activated T cells can also slow down the occurrence of HF to a certain extent. In this study, we found that exercise increased the number of MDSCs in HF mice, so we explored the inhibitory function of MDSCs on T cells. In addition, MDSCs-induced suppression requires cell-to-cell contact through the interaction of cell surface markers and the secretion of temporary mediators.32,33 We isolated and purified MDSCs from isoproterenol group and ET+isoproterenol group, and co-incubated with T cells by cell-cell contact. The proliferation of T cells was analyzed by flow cytometry. Surprisingly, exercise did not increase the inhibitory function of MDSCs on T cells. Furthermore, we also explored the effect of MDSC on cardiac hypertrophy. MDSCs isolated from both isoproterenol and ET+isoproterenol groups could inhibit cardiomyocyte hypertrophy, but there was no difference in their ability to inhibit cardiomyocyte hypertrophy between the 2 groups. Taken together, these results ultimately suggest that exercise can protect the heart by increasing the number of MDSCs in HF mice rather than inhibiting T cells.
Previous studies have shown that circulating levels of inflammatory factors exhibit prognostic importance in the setting of HF.34–37 Proinflammatory cytokines such as IL-6 can recruit and activate MDSCs.38 Consistent with previous studies, our research also found that 1 day and 7 days of isoproterenol treatment can promote the production of proinflammatory cytokine IL-6 in the serum and heart. We also found an increased proportion of MDSC in the heart, bone marrow, peripheral blood, and spleen of mice treated with isoproterenol for 14 days. Therefore, it is unsurprising that the MDSC proportion is positively correlated with levels of these proinflammatory cytokines in mice with HF (Figures 2 and 5), given their role in a compensatory mechanism involved in resolving inflammation. IL-10 plays an important anti-inflammatory effect in many diseases,39–41 and also acts as an important anti-inflammatory cytokine to regulate the fate of MDSC in HF diseases. Our study shows that both 1-day’ and 7-day’ ET can increase the production of IL-10 in serum. IL10/STAT3 signal pathway plays an important protective role in myocardial infarction and ischemia-reperfusion injury.42,43 In this study, we detected the activation of STAT3 in the hearts of isoproterenol and ET+isoproterenol mice. Surprisingly, although there was a slight increase in p-STAT3 levels in ET+isoproterenol mice, it was not statistically significant. Therefore, we detected the expression of p-STAT3 in the nucleus and cytoplasm of each group of MDSCs. The results showed that exercise promoted the transfer of p-STAT3 in MDSCs to the nucleus. In accordance with this, exercise also promoted the transfer of S100A9 in MDSC to the nucleus. Many studies have shown that the accumulation of S100A9 in the nucleus will reduce inflammation and slow down the maturation of MDSCs.21 This suggests that exercise may promote the transfer of S100A9 in MDSCs to the nucleus through the IL10/STAT3 signal pathway, thus enhancing the inhibitory function of MDSCs.
Pervious study confirmed that cardiac macrophages were a dominant source of IL-10 in mice with diastolic.37 In our research, we found that IL-10 was mainly secreted by macrophages in heart of HF mice. To further investigate the mechanism, we bred tamoxifen-inducible Cx3cr1CreER mice with Il10fl/fl mice, hereafter denoted as Cx3cr1-IL10−/−, to obtain mice in which tamoxifen treatment will delete IL-10 in Cx3cr1-expressing monocytes and macrophages. After macrophage-specific knockout of IL-10, ET treatment could not increase the levels of serum IL-10 in HF mice (Figure 7B). However, it was noticed that there was still a certain levels of serum IL-10 in Cx3cr1 IL10−/− mice. This may be due to the fact that IL-10 is a pleiotropic cytokine produced by many kinds of leukocytes including macrophages, DCs, and Th cells.44 In our experiments, ET could significantly increase the expression of IL-10 in WT or IL-10 intact mice but not in Cx3cr1 IL10−/− mice. Those results indicated that macrophages may be the main effector cells in response to ET. Thus, IL-10 knockout in macrophages weaken the protective effect of ET and also reduced the migration of MDSC to the heart, spleen, and peripheral blood mononuclear cells. Interestingly, the knockout of IL-10 in macrophages only changed the number of MDSCs in the body, but not changed their inhibitory function on T cells.
In this study, we found that ET activated the secretion of IL-10 by macrophages in HF mice and promoted the translocation of p-STAT3/S100A9 to the nucleus to regulate the differentiation of MDSCs, thus achieving cardiac protection. Macrophages were a dominant source of IL-10, and thus contributing to ET-induced MDSC expansion in HF mice. MDSCs expansion was not present in macrophage-specific IL-10 knockout mice, and thus the protective effect of ET was abolished in HF mice in accordingly. MDSCs are well known immunosuppressive cells and their immune-regulation effect are associated with iNOS, arginine and an array of immunosuppressive cytokines such as IL-10. Zhou et al16 reported that MDSCs secreted molecules including IL-10 and NO suppressed pressure overload-induced HF in mice. Our experiment also demonstrated that isolated MDSCs could significantly suppress the isoproterenol-induced hypertrophic response in neonatal rat ventricular myocytes. The cytokines secreted by MDSCs in mediating improvement of HF needs to be further investigated. In addition, our study also confirmed that ET can also play a protective role in the treatment of HF. ET improved cardiac function and reduces heart injury caused by HF (Figure 8).
All in all, our research had explored a new theory, proving a new pathway for ET to protect the heart. ET increased the production of IL-10 in macrophages, thereby promoting the nucleation of S100A9 in MDSC, slowing down its maturation process, and increasing the number of MDSC in the body, so as to achieve the protective effect of the heart (Figure 8I). This study had laid a solid theoretical foundation for future clinical applications and provided new ideas for the diagnosis and treatment of HF.
Article Information
Supplemental Material
Methods
Figures S1 and S2
Footnote
Nonstandard Abbreviations and Acronyms
- 5FU
- 5-fluorouracil
- ANF
- atrial natriuretic factor
- EF
- ejection fraction
- ET
- exercise training
- HF
- heart failure
- HW/BW
- heart weight/body weight
- IL
- interleukin
- MDSC
- myeloid-derived suppressor cell
- S100A8
- S100 calcium-binding protein A8
- S100A9
- S100 calcium-binding protein A9
- STAT3
- signal transducer and activator of transcription 3
Supplemental Material
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© 2021 American Heart Association, Inc.
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Received: 9 March 2021
Accepted: 11 November 2021
Published online: 16 December 2021
Published in print: March 2022
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This research was supported by the National Natural Science Foundation of China (Nos. 81971887, 31971194, 82172170, 82072187, and 81802091), the Tianjin Natural Science Foundation (Nos. 20JCYBJC01260, 20JCQNJC01850, 16JCYBJC28100, 18JCQNJC13400, and 17JCQNJC10700), the Technical Envoy for Enterprises of Tianjin Natural Science Foundation (No. 20YDTPJC00250), the Key Laboratory of Emergency and Trauma (Hainan Medical University), the Ministry of Education (No. KLET-201906), and Fundamental Research Funds for the Central Universities (No. 63211140).
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Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.