Interleukin‐7 aggravates myocardial ischaemia/reperfusion injury by regulating macrophage infiltration and polarization

Abstract Interleukin (IL)‐7 is known to enhance the macrophages cytotoxic activity and that macrophages play a pivotal role in the development and progression of myocardial ischaemia/reperfusion (I/R) injury. However, the effects of IL‐7 on macrophages infiltration and polarization in myocardial I/R injury are currently unclear. This study aimed to evaluate the effects of the IL‐7 expression on myocardial I/R injury and their relationship with macrophages. The data showed that IL‐7 expression in mouse heart tissue increases following I/R injury and that IL‐7 knockout or anti‐IL‐7 antibody treatment significantly improve I/R injury, including reduction in myocardial infarction area, a serum troponin T level decreases and an improvement in cardiac function. On the other hand, recombinant IL‐7 (rIL‐7) supplementation induces opposite effects and the anti‐IL‐7 antibody significantly reduces the cardiomyocyte apoptosis and macrophage infiltration. rIL‐7 cannot directly cause apoptosis, but it can induce cardiomyocyte apoptosis through macrophages, in addition to increase the macrophages migration in vitro. Anti‐IL‐7 antibody affects the cytokine production in T helper (Th) 1 and Th2 cells and also promotes the macrophages differentiation to M2 macrophages. However, anti‐IL‐7 antibody does not reduce the M1 macrophage number, and it only increases the ratio of M2/M1 macrophages in mice heart tissues after I/R injury. Taking together, these data reveal that IL‐7 plays an intensifying role in myocardial I/R injury by promoting cardiomyocyte apoptosis through the regulation of macrophage infiltration and polarization.


| INTRODUC TI ON
Coronary artery recanalization, including coronary artery bypass grafting, percutaneous coronary intervention and thrombolytic therapy, is currently the main treatment for myocardial infarction. Coronary artery recanalization is able to clear the narrow or even occluded coronary lumen, but ischaemia-reperfusion (I/R) injury has been the most important obstacle to the blood reflow treatment. 1 Inflammation caused by the infiltration of immune cells, such as macrophages, plays a pivotal role in the development and progression of myocardial I/R injury. 2,3 Interleukin (IL)-7 is produced mainly by thymic stromal cells and can be secreted by other cells, such as foetal liver cells, bone marrow stromal cells, spleen cells, macrophages and dendritic cells. 4 Under pathological conditions, some tumours and endothelial cells can also produce IL-7. 5 Previous research on IL-7 mainly focused on its effects on T and B cells.
IL-7 has been found to be an essential cytokine for growth, survival, differentiation and appreciation of B cells. 6 In addition, these studies found that IL-7 played a key role in the development, proliferation and homeostasis of T cells. 7 However, the other IL-7 functions were also been evaluated by the researchers. Among them, we have paid attention to the fact that IL-7 enhances the macrophage cytotoxic activity, 8,9 induces monocyte macrophages to secrete a variety of pro-inflammatory cytokine factors (MCP-1, MIP, IL-1β, among others), 10 regulates the interaction between various components of the inflammatory process 11 and increases the expression of monocyte-derived inflammatory chemokine receptors (CCR), such as CCR1, CCR2 and CCR5. 12 Monocytes/macrophages are the inflammatory cells with the longest residence time in repairing the myocardial tissue damage after I/R injury. 2,3 In the early stage of inflammation, these cells play a role primarily in the pro-inflammatory response and swallowing and promote the regeneration and remodelling of granulation tissue in the inflammation middle and late stages. 12,13 Importantly, in myocardial I/R injury, inhibiting excessive inflammation can reduce myocardial necrosis, protects vascular endothelial function and reduces infarct area. 14 Besides, it helps to reduce myocardial I/R injury by regulating chemotaxis and macrophages polarization. 15,16 However, it is unknown whether IL-7 can affect myocardial I/R injury by affecting macrophages invasion and polarization.
In this study, we evaluated the IL-7 expression in mice heart tissues following myocardial I/R injury. In addition, we investigated the IL-7 effects on myocardial I/R injury and on chemotaxis and polarization of macrophage by neutralizing endogenous IL-7 and supplementing exogenous IL-7 in mice. The data showed that IL-7 aggravated myocardial ischaemia/reperfusion (I/R) injury by regulating macrophages infiltration and polarization.

| Animals and Myocardial I/R injury in vivo
A total of 81 wild-type (WT) C57BL/6 mice (8-10 weeks) and 6 IL-7 knockout (IL-7-/-) C57BL/6 mice (8-10 weeks) were used in the present study. WT C57BL/6 mice were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). IL-7-/-C57BL/6 mice were generated as previously described. 17 Animal procedures were performed with the consent from the China-Japan Friendship Hospital ethics committee. The I/R mice model was established as described previously. 18 Briefly, mice were anaesthetized with ketamine (50 mg/kg) and pentobarbital sodium (50 mg/ kg). After the mouse thoracic cavity was opened, the left coronary artery (LCA) was quickly found and the ligature was performed (mice in the sham group were not submitted do LCA ligature). The left ventricle was kept ischaemic for 30 minutes because of LCA ligature, which was subsequently removed for reperfusion. The mice were killed after reperfusion for 1, 4, 12, 24, 48 and 72 hours to obtain heart tissues.

| Real-time quantitative PCR
Total RNA in cells and tissues was extracted by an RNA extraction Kit (RC101-01; Vazyme) and was reverse-transcribed into cDNA using the PrimeScript RT reagent (RR047A, Taraka, Japan). Then, 20 μL of RT-qPCR system was prepared as described in the qPCR master mix kit instructions (A6001, Progema, USA). The gene relative expression was calculated by 2 -ΔΔCt method, and β-actin was used as a loading control.
The primers used for qPCR analysis are shown in Table 1.

| Enzyme-linked immunosorbent assay (ELISA) and myeloperoxidase (MPO) activity assay
After 24 hours of reperfusion, venous blood from mice was collected through the posterior orbital venous plexus to determine the serum troponin T level using an ELISA kit for troponin T (Mito Sciences).

| Endogenous IL-7 neutralization and exogenous rIL-7 repletion
Thirty minutes of ischaemia and 24 hours of reperfusion were chosen as treatment time-points. The mice were randomly divided into sham, IgG, anti-IL-7, vehicle and rIL-7 groups (eight mice in each group). In the sham group, the mice only had their thorax open, were not submitted to LCA ligature and received no treatment. In the IgG group, the mice were injected IV with 100 μg rat IgG2A isotype control (MAB006; R&D). In the anti-IL-7 group, the mice were injected IV with 100 μg mouse IL-7 monoclonal antibody (MAB4071, R&D).
In the vehicle group, the mice were injected IV with 50 μL sterile saline. Finally, in the rIL-7 group, the mice were injected IV with 1μg IL7 recombinant mouse protein diluted in 50 μL sterile saline.
After 25 minutes of ischaemia, mice in the IgG, Anti-IL-7, Vehicle and rIL-7 groups were injected IV with the corresponding reagent and continued to perform ischaemia for 5 minutes and reperfusion for 24 hours.

| Cardiac function and haemodynamic analysis
An animal electrocardiogram (ECG) record analyser (LS20, B&E TEKSYSTEMS, China) to measure the mice ECG for later calculation of the ejection fraction and fraction shortening of left ventricular (LV), LV end-diastolic pressure (LVEDP) and maximal (LV + dp/dt max ) according to the standard formulas. 19

| Myocardial infarction area measurement
The killed mice hearts were harvest, and the myocardial infarction area was detected using TTC staining. The preparation of heart tissue sections and TTC staining were performed as described previously. 20 Image J software (National Institutes of Healt) was used to the measure the area of myocardial infarction per section. The myocardial infarction volume was calculated by multiplying the myocardial infarction area in each slice by the section thickness (2 mm).

| Apoptosis analysis
TUNEL staining was used to determine the apoptosis level in the heart tissues as described previously. 18 Briefly, the mice hearts were fixed with paraformaldehyde, embedded in paraffin and sliced into tissue sections. After dewaxing and hydration, the tissue sections were incubated with proteinase K to permeate the cells. Then, the sections were incubated with TUNEL reaction solution for 1 hour at 37°C followed by colour development with DAB solution. All reagents and procedures for the TUNEL assay came from the TUNEL Cell Apoptosis Detection Kit (TA201-02; TRANSGEN). Moreover, a Caspase 3 activity assay kit (C1115; Beyotime) was used to measure the Caspase 3 activity in vitro following the manufacturer's instructions. The cells were harvested, washed with cold PBS and fixed with cold 70% ethanol at 4°C for 1 hour. Then, the cells were resuspended using cold PBS after removing the ethanol by centrifugation. As stated by the manufacturer's instructions (E606336, Sangon Biotech), 15 minutes after adding the labelling reagent, the cells were collected and resuspended with PBS to be detected. Finally, the apoptosis level was also assessed by measuring Bax and Bcl2 expression by Western blot.

| Mouse neonatal cardiomyocytes isolation
Neonatal mice (1 day old) were killed by cervical dislocation to collect the heart tissues as previously described. 21 Next, the heart tissues were cut into small pieces and incubated with trypsin/EDTA solution (25200056; Thermo Fisher) for 30 minutes at 4°C. Then, the tissues were washed and the incubation was stopped by adding equal volume of DMEM medium (10567022; Gbico) containing 20% foetal bovine serum (FBS; 16140071, Gbico). After centrifugation, the tissues were collected and incubated with Liberase TH (5401151001; Roche) for 15 minutes at 37°C. The incubation with Liberase TH was repeated to ensure that all myocardial cells were harvest, which were subsequently filtered using a nylon cell strainer.

| Treatment of mouse neonatal cardiomyocytes
After 72 hours of culturing neonatal mouse cardiomyocytes, 1 × 10 6 of these cells were seeded in the lower chamber of the polycarbonate insert cell culture device (140640, Thermo Fisher, USA).
Subsequently, different concentrations of rIL-7 were added to the culture medium for 24 hours and then the cells were collected for apoptosis evaluation.
In a co-culture system of macrophages and cardiomyocytes, 1 × 10 6 macrophages were seeded in the upper chamber of polycarbonate insert cell culture device and 1 × 10 6 neonatal cardiomyocytes were seeded in the lower chamber of that same device.
Subsequently, different concentrations of rIL-7 were added to the culture medium for 24 hours and then the cells were harvested for apoptosis analysis.
In a separate macrophage culture system, 1 × 10 6 macrophages were seeded in the upper chamber of polycarbonate insert cell culture device. Next, the conditioned medium (CM) of macrophages was collected after stimulation with 50 ng/mL rIL-7 for 24 hours. In a separate cardiomyocyte culture system, 1 × 10 6 neonatal cardiomyocytes were seeded in the lower chamber of polycarbonate insert cell culture device and the conditioned medium (CM) described above was used to culture the cardiomyocytes for 24 hours. Finally, 24 hours after stimulation with or without 100 μmol/L H 2 O 2 , the cells were collected for apoptosis assessment.

| Macrophages migration assay
Mouse primary peritoneal macrophages were prepared and cultured according to previously described methods. 22  and incubated for 24 hours at 37°C. In the rIL-7 and anti-IL-7R groups, 10 mg/mL CD127 monoclonal antibody was first added and incubated for 2 hours, and then, 50 ng/mL rIL-7 was added and incubated for 24 hours. The cells were collected from the lower chamber, and the culture medium was removed. Then, they were washed three times with PBS, stained with 0.25% crystal violet for 25 minutes, slowly rinsed with sterile water, place on a sterile ultra-clean bench to dry and finally the absorbance at 595 nm was measured.

| Heart infiltrating cell isolation and flow cytometry analysis
Cardiac single cell suspensions were prepared as previously described. 23  and iNOS+ cells (M1 macrophages) was analysed in this group of cells.

| Statistical analysis
The data were analysed by Graphad prism software (v. 8

| IL-7 increases after myocardial I/R injury
The investigation of the IL-7 involvement in myocardial I/R injury was initiated by the detection of dynamic changes in the IL-7 mRNA expression using RT-qPCR in mice heart tissues at 1, 4, 12, 24, 48 and 72 hours after reperfusion. As showed in Figure 1A

| IL-7 aggravates myocardial I/R injury
To study the effect of IL-7 expression on the development of myocardial I/R injury, the myocardial I/R injury was compared between wild-type (WT) C57BL/6 mice and IL-7 knockout (IL-7-/-) mice based on wild-type C57 BL/6 mice by determining the infract size of left ventricular (LV) (Figure 2A,B), the serum troponin T level ( Figure 2C), the ejection fraction ( Figure 2D), the fraction shortening ( Figure 2E), the left ventricular end-diastolic pressure (LVEDP) ( Figure 2F) and the maximum systolic blood pressure (dP/dt max ) ( Figure 2G). The results showed that the LV infract size, serum troponin T level and LVEDP in IL-7-/-mice were all significantly lower than in WT mice. In contrast, the ejection fraction, fraction shortening and dP/dt max in IL-7-/-mice were all significantly higher than in WT mice.
To determine whether IL-7 could be a potential target for the myocardial I/R injury treatment, myocardial I/R mice were treated systemically with neutralizing anti-IL-7 monoclonal antibody (mAb) or recombinant IL-7 (rIL-7) before reperfusion. Myocardial injury markers and cardiac function were measured 24 hours after reperfusion because the IL-7 expression peaked in this time-point after reperfusion. As shown in Figure 3A, treatment with neutralizing anti-IL-7 antibody led to a significantly decrease in the LV infract size ratio, whereas this ratio increased significantly after exogenous rIL-7 repletion. Meanwhile, the levels of myocardial injury biomarkers and serum troponin T ( Figure 3B) increased significantly after I/R injury. Neutralizing anti-IL-7 antibody treatment significantly reduced these levels, whereas exogenous rIL-7 repletion increased them significantly. To study the IL-7 effects on cardiac function following I/R injury, ejection fraction ( Figure 3C), fraction shortening ( Figure 3D), LVEDP ( Figure 3E) and dP/dt max ( Figure 3F) were also evaluated. The data showed that the ejection fraction, fraction  6). Comparison between the two groups, *** was P < .001. Scale bar = 1 cm shortening and dP/dt max were all significantly decreased after I/R injury, whereas LVEDP was significantly increased. Importantly, neutralizing anti-IL-7 antibody administration was able to reverse these changes, whereas exogenous rIL-7 repletion significantly enhanced them.

| IL-7 promotes I/R-induced cardiomyocyte apoptosis
Apoptosis contributes greatly to I/R injury and is a direct factor that causes changes in cardiac function and myocardial injury. 24 Therefore, to study the IL-7 mechanism in the I/R injury, apoptosis in the LV section of heart tissues 24 hours following I/R injury was assessed using TdT-mediated dUTP Nick-End Labeling (TUNEL) staining. In this study, the apoptosis cells showed brown staining ( Figure 4A). The apoptosis cell number was significantly increased fol- After 24 hours of incubation, cardiomyocyte apoptosis was evaluated. As shown in Figure 5C, rIL-7 induced cardiomyocyte apoptosis in a dose-dependent manner. Furthermore, a macrophage conditioned medium (CM) was prepared by adding 50 ng/mL rIL-7 into macrophages medium. CM was used to culture cardiomyocytes and was able to further increase their apoptosis ( Figure 5D).

| IL-7 increases macrophage infiltration into heart tissues following I/R
Macrophage infiltration is a hallmark pathological change in early stage of I/R injury and one of the main causes of myocardial dam- The rIL-7 effect on the ability of macrophages to migrate was evaluated in vitro and revealed that rIL-7 promoted macrophage migration in a dose-dependent manner ( Figure 7A). Then, the rIL-7 mechanism of promoting macrophage migration was analysed by neutralizing IL-7R antibody treatment. The data showed that, although the use of IL-7R neutralizing antibodies alone did not affect macrophage migration, IL-7R neutralizing antibodies were able to significantly reduce the rIL-7-induced high macrophage migration ( Figure 7B). Therefore, these data suggested that IL-7 promoted macrophage migration via targeting IL-7R.

| IL-7 alters T helper (Th)1 and Th 2 cytokines following I/R injury in vivo
To study the IL-7 expression effects on the acute inflammatory response after I/R injury, the mRNA levels of IFNγ and TNFα (Th1 cytokines) and IL-4 and IL-13 (Th2 cytokines) were determined using RT-qPCR. In addition, protein levels of IFNγ, TNFα, IL-4 and IL-13 were assessed by ELISA (Figure 8). The use of IL-7 neutralizing antibody led to a reduction in the IFNγ and TNFα mRNA levels, and an F I G U R E 5 IL-7-induced cardiomyocyte apoptosis requires mediation of macrophages. (A) Western blot analysis of the indicated IL-7 receptor (IL-7R) protein levels in mouse peritoneal macrophages and mouse neonatal cardiomyocyte; (B, C) Mouse neonatal cardiomyocyte apoptosis under different doses of IL-7 in a separate culture system for cardiomyocytes (B), or in a co-culture system of macrophages and cardiomyocytes (C); (D) The conditioned medium was obtained by adding 50 ng/mL rIL-7 to the macrophage culture medium for 24 h, and then using this conditioned medium to culture the mouse neonatal cardiomyocyte. 24 h after adding 100 μmol/L H 2 O 2 , mouse neonatal cardiomyocyte was harvested and analysed for apoptosis. Data shown are mean ± SD, and 3 independent replicates for each experiment. ns was P > .05, # was P < .05 and ### was P < .001 versus Control group. Comparison between the two groups, *** was P < .001 increase in the IL-4 and IL-13 levels in heart tissues following I/R injury 24 hours after reperfusion ( Figure 7A-D). In contrast, exogenous rIL-7 repletion markedly increased the IFNγ and TNFα mRNA levels and decreased the IL-4 and IL-13 levels. Remarkably, the results observed in the mRNA level evaluation were also observed in the assessment of IFNγ, TNFα, IL-4 and IL-13 protein levels ( Figure 7E-H).  Figure 9H). This evidence show that IL-7 can promote macrophage differentiation into M1 macrophages and decrease the M2/M1 macrophage ratio in heart tissues after I/R injury.

| DISCUSS ION
In the present study, we revealed the important role of IL-7 in me- patients. 39,40 Here, we found that IL-7 expression increased rapidly in the early post-reperfusion period and peaked in 24 hours, which suggests that IL-7 may be involved in the myocardial I/R injury in the early period.
To investigate the IL-7 mechanism in the myocardial I/R injury, we measured apoptosis in heart tissues after I/R injury. Although there are many mechanisms that cause I/R injury, such as inflammation, oxidative stress and mitochondrial damage, cardiomyocyte apoptosis is a direct I/R injury physiological manifestation. 41,42 The data from the present study showed that endogenous IL-7 neutralization reduced apoptosis and apoptosis-related marker levels in heart tissues following I/R injury. This results in the amplification of the inflammatory cascade in the heart, thereby exacerbating myocardial I/R injury. 12,13 In this study, we found that IL-7 neutralization can significantly reduce the macrophage infiltration in cardiac tissues following I/R injury in vivo, as well as we found that rIL-7 promoted macrophage migration in vitro.
Therefore, IL-7 exacerbates myocardial I/R injury by activating macrophages and promoting macrophage migration to damaged tissues.
Macrophages have two phenotypes, namely M1 and M2. M1 macrophages have iNOS as their specific marker and can promote in balance, whereas this balance is broken in damaged tissues and organs, such as in heart tissue following by I/R. 30,31 In the process of ischaemia-reperfusion injury, neutrophils bind with adhesion factors on vascular endothelial cells and cardiomyocytes to release many inflammatory/anti-inflammatory mediators with chemotactic effects. 12 Therefore, in this study, pro-inflammatory mediators play a major role in the heart tissue after I/R injury without intervention, so macrophages are polarized to M1 macrophages. In addition, cytokines secreted by T helper cells (Th) have macrophage polarization as their main role. According to the difference in secreted cytokines, Th cells are divided into two types, Th1 and Th2. Th1 cells are characterized by secreting cytokines, such as interferon Y, IL-1, IL-2 and TNF-a, which polarizes macrophages into M1 macrophages.
Th2 cells secrete IL-4, IL-5, IL-6, IL-10 and IL-13, which polarizes macrophages into M2 macrophages. 47,48 Based on these data, we investigated the expression of some markers related to macrophage polarization and phenotype. We found that the endogenous IL-7 neutralization increased the ratio of M2/M1 macrophages, whereas the exogenous IL-7 repletion decreased it in heart tissues following I/R injury. The data showed that IL-7 promoted polarization of M2 macrophages.
In conclusion, the data from this study suggest that IL-7 is produced rapidly in heart tissues during early ischaemia-reperfusion and aggravates myocardial I/R injury by regulating macrophage infiltration and polarization. Our study also shows that the control of Comparison between the two groups, *** was P < .001 endogenous IL-7 levels immediately after ischaemia-reperfusion can help to reduce myocardial I/R injury.

ACK N OWLED G EM ENT
We are appreciative to Wei Gong for his help in the research.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.