Interleukin‐38 alleviates cardiac remodelling after myocardial infarction

Abstract Excessive immune‐mediated inflammatory reaction plays a deleterious role in ventricular remodelling after myocardial infarction (MI). Interleukin (IL)‐38 is a newly characterized cytokine of the IL‐1 family and has been reported to exert a protective effect in some autoimmune diseases. However, its role in cardiac remodelling post‐MI remains unknown. In this study, we found that the expression of IL‐38 was increased in infarcted heart after MI induced in C57BL/6 mice by permanent ligation of the left anterior descending artery. In addition, our data showed that ventricular remodelling after MI was significantly ameliorated after recombinant IL‐38 injection in mice. This amelioration was demonstrated by better cardiac function, restricted inflammatory response, attenuated myocardial injury and decreased myocardial fibrosis. Our results in vitro revealed that IL‐38 affects the phenotype of dendritic cells (DCs) and IL‐38 plus troponin I (TNI)‐treated tolerogenic DCs dampened adaptive immune response when co‐cultured with CD4+T cells. In conclusion, IL‐38 plays a protective effect in ventricular remodelling post‐MI, one possibility by influencing DCs to attenuate inflammatory response. Therefore, targeting IL‐38 may hold a new therapeutic potential in treating MI.

IL-38 is located in the IL-1 family cluster on chromosome 2 and encoded by IL1F10. 12,13 As with other members of IL-1 family, it lacks a signal peptide and caspase-1 consensus cleavage site. IL-38 plays biological effects through acting on IL-1 receptor antagonist (IL-1Ra) and IL-36 receptor antagonist (IL-36Ra). [13][14][15] Many studies have been reported that IL-38 gene polymorphisms are associated with many inflammatory diseases, such as rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis. [16][17][18] In addition, the expression levels of IL-17 and IL-22 can be suppressed by IL-38 in candida albicansstimulated peripheral blood mononuclear cells (PBMCs). 14 Moreover, depletion of IL-38 in cultured apoptotic macrophages exacerbated the expression of IL-6 and IL-8. 19 We have previously shown that plasma IL-38 level in ST-segment elevation myocardial infarction (STEMI) patients was positively correlated with CRP, cTNI and NT-proBNP, but was weakly negatively correlated with left ventricular ejection fraction (LVEF). 20 Our data indicated that IL-38 appears to be a potentially novel biomarker for patients with STEMI. However, its exact biological effect in MI needs to be further elucidated.
In this study, we aim to clarify the effect and mechanism of IL-38 in post-MI remodelling. Our results indicated that rIL-38 could ameliorate cardiac remodelling and improve cardiac function in post-MI mice. In addition, IL-38 imposed a regulatory phenotype on DCs and inhibited inflammatory factors secretion in vitro. Our data indicated that IL-38 could induce a tolerogenic immune response and ameliorate post-MI remodelling in mice.

| Animals
Male C57BL/6 mice used in this study were purchased from Beijing HFK Bioscience Co., Ltd (Beijing, China) and maintained in Tongji Medical College Animal Care Facility on a chow diet according to institutional guidelines. Experiments involving mice (per cage n = [5][6] were given with normal food and water prior to experiments and kept under standard animal room conditions (temperature, 21±1˚C; humidity, 50-60%; 0.03% CO2; 12 hours for light and 12 hours for dark). All studies were approved by the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology, and the experimental methods were performed in accordance with the approved guidelines.

| Surgical protocol
MI model was induced by permanently ligating the left anterior descending (LAD) coronary artery. In brief, after anaesthesia with intraperitoneal injection of sodium pentobarbital (60 mg/kg, 1%, 5 μl/g), mice were intubated and ventilated through a rodent respirator. Adequate anaesthesia was assured by the absence of reflexes prior to surgery. Chest was opened in the third or fourth intercostal space and LAD coronary artery of the heart was ligated with a 7-0 prolene suture. Mice in the sham-operated group were subjected to identical operation, but without the LAD coronary artery ligation.

| Echocardiography
The cardiac function of mice at one and four weeks post-MI was evaluated non-invasively by echocardiography performed with Vevo1100 (Visualsonics, Toronto, Canada) equipped with a 30 MHz transducer-phased-array transducer. Mice were anaesthetized with sodium pentobarbital and two-dimensional echocardiographic views of the mid-ventricular short axis and parasternal long axes were obtained. Left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), ejection fraction (EF) and fractional shortening (FS) were calculated from the digital images using a standard formula as previously described. 21 The sonographer was blinded to the randomization of mice.

| Dendritic cell
Bone marrow-derived dendritic cells (BMDCs) were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) (40 ng/mL) and IL-4 (20 ng/mL). 6-week-old male C57BL/6 mice were used to acquire bone marrow in this study. Bone marrow cells were depleted of red blood cells and washed with phosphate-buffered saline (PBS), then resuspended at a density of 2 × 10 6 cells/mL in complete culture medium (RPMI-1640 suspended with 10% heatinactivated foetal calf serum, GIBCO, Carlsbad, CA) with 40 ng/mL recombinant mouse GM-CSF (Peprotech, Rocky Hill, NJ) and 20 ng/ mL IL-4 (Peprotech) at 37°C with 5.0% CO2. Half of the culture medium was replaced with the same concentrations of GM-CSF and IL-4 every two days. Immature dendritic cells (imDCs) were obtained after 6-8 days of culture. Magnetic cell-sorting kit of CD11c (Miltenyi Biotec, Auburn, CA) was used to purify DCs from the differentiated bone marrow cells. Different DC subsets were treated as follows: (i) imDCs: No additional incubation; (ii) mDCs: CD11c + imDCs were incubated with 100 ng/mL lipopolysaccharide (LPS) for 24 hours; (iii) IL-38-LPS-DCs: CD11c + imDCs were incubated with 100 ng/mL LPS and 50 ng/mL IL-38. All the DCs were incubated in complete culture medium.

| Preparation of splenic CD4 + cells
Spleens were removed from anaesthetized mice, mushed and then passed through a 100-µm nylon mesh in PBS to remove connective tissue. Erythrocytes were excluded and the splenic mononuclear cells were collected with lymphocyte separation fluid (MP Biomedicals, USA). 22 For the separation of CD4 + cells, magnetic cell-sorting kit of CD4 (Miltenyi Biotec, Auburn, CA) was used to purify CD4 + cells from the differentiated splenic cells according to the manufacturer's instructions.

| Cardiomyocytes
Cardiomyocytes required for in vitro experiments were isolated from hearts of 1-to 3-day-old mouse. Hearts were digested in 0.05% Trypsin-EDTA (Gibco) for about 30 minutes on ice followed by serial digestions in collagenase type II (Worthington) at 37˚C and preplated twice in T75 culture flasks (Sarstedt) for removing fibroblasts.

| Isolation of infiltrating DCs from the infarcted heart
On the indicated days after the operation, mice were deeply anaesthetized. Mice hearts were obtained after intracardially perfusing with PBS to remove blood cells before euthanasia. Infarcted heart tissue was acquired after dissecting using fine scissors, and minced infarcted heart tissue was enzymatically digested at 37°C with a cocktail of type II collagenase (Roche Diagnostics; 1 mg/mL in HEPES buffer). Isolated cell suspensions were filtered with a 100-μm cell strainer after digestion, then suspended in RPMI-1640 containing 3 % FCS. Cardiac infiltrating DCs were isolated through density gradient centrifugation. For the separation of dendritic cells, cells were incubated with CD45-PE-cy7, CD11b-FITC and CD11c-APC for 30 minutes.

| Real-time PCR analysis
Total RNA was extracted from cultured cells or tissues using Trizol (Invitrogen, Carlsbad, CA) and converted into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Dalian, China).

SYBR Green Master Mix (Takara Biotechnology, Dalian, China)
was used to quantify mRNA levels of target genes with an Applied Biosystems 7500 Real-Time PCR system (BIO-RAD, Singapore). mRNA expression level of each sample was normalized to that of GAPDH. Primer sequences used in this study are listed in Table 1.

| Western blot analysis
Total protein was extracted from heart tissues using RIPA lysis buffer (Beyotime) and quantified using a BCA protein assay kit (Pierce Biotechnology Inc). The following primary antibodies were used: anti-rat GAPDH (Immunoway, USA) and anti-mouse IL-38 (R&D Systems, USA). Protein samples were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes.
After being blocked with 5% defatted milk for 1-2 hours at room temperature, the membranes were incubated with the appropriate primary antibodies and incubated at 4°C overnight, followed by incubation with an HRP-conjugated secondary antibody. The specific bands were visualized using the Super ECL reagent (Thermo Scientific). ImageLab 3.0 software (Bio-Rad Laboratories, Hercules, CA) was used for analysis.

| Immunohistochemistry and immunofluorescence
Selected heart tissues were embedded in paraffin and used for immunohistochemical studies. For the assessments of inflammatory cell areas in damaged heart, heart sections (5 μm) were stained with haematoxylin-eosin (HE). For the analysis of collagen volume fraction, heart sections(5 μm) were stained with Masson's trichrome. (1:100, Abcam, UK) to identify macrophages and cardiomyocytes.

| Statistical analysis
All data are shown as means ± SEM. Differences between 2 groups and among multiple comparisons were analysed respectively using unpaired Student's test and one-way ANOVA, followed by Bonferroni's test. Survival rate was analysed by Kaplan-Meier survival analysis and compared by the log-rank test. GraphPad Prism 6.0 (GraphPad Software, Inc, CA) was used for all the analyses, and P < .05 was considered statistically significant.

| Cardiomyocytes are responsible for producing IL-38
To

| The role of IL-38 on survival and cardiac function
The As shown in Figure 3B-F, compared with PBS-treated mice, echocardiographic assessment consistently revealed that EF and FS were greater in IL-38-treated mice on day 7 and day 28 post-MI, LVEDD and LVESD was smaller on day 28 after MI. Thus, it seems that cardiac function was significantly prevented in the IL-38-treated group after MI.

| IL-38 inhibits cardiomyocyte apoptosis and alleviates cardiac fibrosis
IL-38 has been shown to promote heart function in post-MI mice, we next tried to determine the mechanism underlying how IL-38  Dead myocardium is replaced by non-contractile fibrous scar tissue that leads to ventricular dysfunction. 24 We therefore analysed fibrosis on day 7 in the heart. As shown in Figure 4D,E, IL-38-treated mice exhibited markedly reduced fibrotic areas than PBS-treated mice.

| IL-38 inhibits inflammatory response in infarcted heart
In the previous steps of the experiment, we found that IL-38-treated mice showed less cardiomyocyte apoptosis and cardiac fibrosis, so we tried to investigate whether the beneficial effects of IL-38 are associated with the suppressed inflammatory responses. As shown in Figure 5A,B, infiltration of inflammatory cells was softer in IL-38treated mice compared with PBS-treated mice reflected by immunohistochemical staining with HE on day 3. Neutrophils are rapidly recruited in the event of heart damage, followed by proinflammatory monocytes and lymphocytes. 8 We next tested infiltration of neutrophils on day 3 and macrophages on day 7 in damaged hearts. Data showed that the number of neutrophils that infiltrated into the infarcted myocardium was obviously lower in IL-38-treated mice than that in control mice, and the difference of macrophages infiltration degrees between the two groups was in line with this result (Figure 5A,B).
In the complex environment of infarcted heart, malignant inflammatory response may be somewhat prolonged due to the effects of a variety of cytokines. We next measured the spatially and temporally expression of cytokines in the infarcted heart. As shown in Figure 5C-F, the levels of IL-6, tumour necrosis factor (TNF)-α, IL-1β and IL-17A were reduced in IL-38-treated mice than in control mice with MI.

| The role of IL-38 on the phenotype of DCs
DCs act as an effective immune regulator during the post-infarction healing process via its regulation of immune cells homeostasis. 25 In addition, high expression of IL-36R, the membrane receptor for IL-36, IL-36Ra and IL-38, was detected on DCs. 14

| IL-38 plus TNI-treated DCs exhibit more tolerogenic properties
Cardiac troponin I (cTNI) is a gold standard biomarker for the acute coronary syndrome, as it indicates myocardial cell damage with high sensitivity and specificity. 26 TNI-pulsed DCs can induce antigen-specific immune responses. We used low-dose LPS and TNI for inducing antigen-specific tolerogenic DCs (tDCs). As shown in Figure 7A-E, DCs pulsed with IL-38 and TNI showed higher expressions levels of IDO and IL-10 and lower expression levels of IL-23, TNF-α and IFN-γ than TNI-loaded DCs.

| IL-38-tDCs augment the percentage of regulatory T cells in vitro
CD4 + T cell activation and homeostasis are involved in facilitating wound healing after MI. 27 Next, a series of functional co-culture experiments were performed to investigate the role of IL-38-treated tDCs on CD4 + T cells. As shown in Figure 7F

| D ISCUSS I ON
Previously, our clinical data indicated that circulating IL-38 is a potentially novel biomarker for in STEMI patients. 20 In the present study, we further investigated the role of rIL-38 in ventricular remodelling after MI. We found that MI induced IL-38 expression and rIL-38-treated mice showed better cardiac function and less myocar-  33 We previously reported that circulating IL-38 was increased in STEMI patients, which indicated that IL-38 may act as a promotive role in the development of MI. 20 In this study, we ob- IL-38 expression has been reported in many organs and tissues, such as skin, tonsil, thymus, spleen, foetal liver and salivary glands. 36 It has been recently found that IL-38 can be released from apoptotic cells to limit inflammatory response induced by macrophage. 19 However, few reports of its expression or function in heart disease have been reported. Whether intrinsic cardiac cells or circulating and/or homing extracardiac cells were responsible for producing IL-38 was not known. In this study, we discovered that IL-38 was increased, especially in peri-infarct zone of mice heart with MI. IL-38 was mainly expressed in cardiomyocytes in the process of MI, though it was even detected in CD68 + lesional macrophages at 7 days after MI. We also found a similar effect in cultured cardiomyocytes exposed to exogenous H 2 O 2 oxidative stress. Overexpression of IL-38 in cultured cardiomyocytes reduced the proto anti-apoptotic ratio of Bcl-2 family proteins, which is an upstream regulator of mitochondrial cytochrome c release.
The ratio of Bax to Bcl-2 is an important determinant of the cell's susceptibility to undergo apoptosis. 37 Our data indicated that in- DCs are tolerogenic, whereas fully mature DCs are immunogenic. 38,39 Normally, tolerogenic DCs play critical roles in inducing peripheral tolerance by suppressing effector T cells, activating regulatory (Treg) cells and negative modulating Th1/Th2 immune responses. 40,41 Interestingly, Toshihisa et al have reported that DCs act as a potent immune protective regulator via its control of monocyte/macrophage homeostasis in the post-infarction healing process. 25 In addition, high expression of IL-36R, the membrane receptor for IL-36, IL-36Ra and IL-38, was detected on DCs. 14  So far, the receptors for IL-38 have not been fully established.
IL-38 might act as an IL-1 family antagonist for the highly homologous toIL-36Ra and IL-1Ra. 31 It has been shown that IL-38 binds to IL-36R and neutralizes the IL-36 cytokine signalling to exert anti-inflammatory effects. 14 However, recently study reported that IL-38 can bind toIL-1RAPL1 to limit cytokine production in a broader inflammatory context. 19 Here, we showed that IL-38 can exert anti-inflammatory effects post-MI via affecting the phenotype of DC, suggesting that IL-38 could target DCs in addition to macrophages. However, according to the present results, it is difficult to distinguish the underlying mechanism for the beneficial effect of IL-38. We need IL-38 knockout mice and specific DCs knockout mice for further mechanism study. Therefore, the molecular mode of action and receptor signalling pathway of IL-38 in MI need to be further investigated.
In conclusion, we demonstrated that IL-38 plays a protective effect in ventricular remodelling post-MI in this study, one possibility by influencing the regulatory function of DCs to attenuate inflammatory response. However, relatively small sample size and unclear signal mechanism of IL-38 should be considered in the study.
Whether IL-38 should be considered as a new therapeutic molecule in MI deserves further experiments.

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

DATA AVA I L A B I L I T Y S TAT E M E N T
The data can be available and cited.

R E FE R E N C E S
1. Parikh NI, Gona P, Larson MG, et al. Long-term trends in myocardial infarction incidence and case fatality in the National Heart,