The role of dendritic cells regulated by HMGB1/TLR4 signalling pathway in myocardial ischaemia reperfusion injury

Abstract Inflammatory response plays an important role in ischaemia reperfusion injury (IRI) through a variety of inflammatory cells. Apart from neutrophils, macrophages and lymphocytes, the role of dendritic cells (DCs) in IRI has been noticed. The study was aimed at investigating whether the high‐mobility group protein box‐1/toll like receptor 4 (HMGB1/TLR4) signalling pathway regulate the migration, adhesion and aggregation of DCs to the myocardium, induce DCs activation and maturation, stimulate the expression of surface costimulatory molecules and participate in myocardial IRI. In vivo, migration, adhesion, and aggregation of DCs was enhanced; the expression of peripheral blood DCs CD80 and CD86, myocardial adhesion molecules were increased; and the infarct size was increased during myocardial ischaemia reperfusion injury myocardial ischemic/reperfusion injury (MI/RI). These responses induced by MI/RI were significantly inhibited by HMGB1 specific neutralizing antibody treatment. Cellular experiments confirmed that HMGB1 promoted the release of inflammatory cytokines through TLR4/MyD88/NF‐κB, upregulated CD80 and CD86 expression, mediated the damage of cardiomyocytes and accelerated the apoptosis. Our results indicate that DCs activation and maturation, stimulate the expression of surface costimulatory molecules by promoting the release of inflammatory factors through NF‐κB pathway and participate in myocardial IRI.


| INTRODUC TI ON
Over the past decades, with the development of thrombolytic therapy, cardiovascular surgery under cardiopulmonary bypass and percutaneous transluminal coronary angioplasty, the survival rate of heart disease patients has been significantly improved.
However, myocardial ischaemia reperfusion injury myocardial ischemic/reperfusion injury (MI/RI) remains a major obstacle to the treatment of cardiovascular disease. [1][2][3] Reducing MI/RI has been a hot research topic in cardiovascular field. The pathogenesis of MI/RI is complex, among which local and systemic inflammatory response is the most prominent characteristic. 4 The pathogenesis involves the infiltration of inflammatory cells and the production of inflammatory cytokines and chemokines, which leads to substantial damage of myocardium. Apart from neutrophils, macrophages and lymphocytes, the role of dendritic cells (DCs) in ischaemia reperfusion injury (IRI) has been recently received considerable attention by researchers. [5][6][7] Dendritic cells is a critical component of innate immune and adaptive immune response. It is one of the key inflammatory cells that affect the development and progression of IRI, however, the mechanism involved is perplexing. Literature has emerged that offers contradictory findings, and there is no general agreement on the role of DCs in IRI to date. This is due to its role in regulating immune function, which is closely related to the maturation, activation degree, quantity and local microenvironment of DCs. [8][9][10] In addition, little is known about its function, occurrence, and interaction between groups. A considerable amount of literature has been published about liver and kidney IRI, however, no previous study has investigated that which signalling pathway mediates DCs in the MI/RI process, so the role of DCs in the pathogenesis of MI/RI needs more researches to explore. 11 High-mobility group protein box-1 (HMGB1) is a specific ligand of toll like receptor 4 (TLR4) and its release into extracellular space or serum after cell necrosis or injury produces a wide range of cellular biological effects, [12][13][14] while TLR4 is mainly expressed on macrophages, DCs and other cells. 15 In recent years, studies have confirmed that HMGB1/TLR4 signalling pathway plays an important role in IRI. [16][17][18] However, it's still not clearly defined whether or not HMGB1 controls the function of DCs through the TLR4 on the DCs, thus affecting the role of DCs in MI/RI. This study is based on rat model of MI/RI, DCs intervention experiment, DCs and myocardial cell co-culture experiment, HMGB1, HMGB1 antibody and TLR4 antagonists treatment, to explore the effect of HMGB1/TLR4 pathway on DCs function and its mechanism, to provide ideas and theoretical basis for the prevention and treatment of MI/RI.

| Animals
Male Sprague-Dawley (SD) rats (6)(7)(8)  in the controlled environment of the animal center at 25 ± 1°C under a 12 hour light-dark cycle, and they were allowed free access to food and water.

| Rat model of MI/RI
Rats were anesthetized by inhaling isoflurane. Peripheral blood was collected by femoral venipuncture. Then the animals were intubated for artificial ventilation with 100% oxygen using a breathing machine (tidal volume 5 mL, frequency 80 per minute) and monitored by Electrocardiogram. Thoracotomy was performed between the sternum and left costa, and then the pericardium was opened.
Myocardial ischaemia was induced by ligating the left anterior descending coronary artery (LAD) using a 3-0 silk suture, and the coronary artery was occluded by pulling on the suture tightly 10 minutes later. After 30 minutes of myocardial ischaemia, reperfusion started by releasing the ligature and removing the tube for 180 minutes. The sham group were anaesthetized and their LAD was threaded but not ligation.
The indications of successful LAD occlusion or reperfusion included ECG and myocardial color. 19

| Isolation, culture, purification and identification of cardiac myocytes in rat
See the Supplementary Materials and Methods for details.

| Experimental grouping and processing of in vivo experiments
As shown in the Figure 1, SD rats were divided into four groups ran- were divided into four groups randomly, with six rats in each group.
The experimental grouping was the same as above, and the range of myocardial infarction was measured.

| Blood collection and tissue harvest
After 180 minutes of reperfusion (group Sham at the corresponding time point), the blood was collected immediately through the femoral vein and used for the detection of inflammatory factors and DCs costimulatory molecules. After collecting the blood, the rat heart was obtained and partial myocardial tissue was fixed with 4% polyoxymethylene, paraffin embedded for pathology and histochemical examination; additional myocardial tissue for protein and gene detection. The myocardial infarct size was assessed by Evan's Blue and 2,3,5-triphenyl tetrazolium chloride (TTC) double staining method.

| Expression of pbDCs costimulatory molecules
After the pbDCs were isolated, cultured and purified, they were incubated with CD11c antibody, then the flow cytometry detection was applied to determine the purity of the DCs. If the purity of cells were higher than 90%, the cells were sampled by flow cytometry and incubated with CD86 (sc-28347; Santa Cruz), then the CD86 signal intensity was detected by flow cytometry. After incubated with CD80 antibody (sc-58911; Santa Cruz), then added Fluorescein (FITC) -conjugated Affinipure Goat Anti-Rat IgG (SA00003-11; Proteintech, Rosemont, IL) to incubate. Detecting the signal intensity of CD80 by flow cytometry.

| Hematoxylin-eosin staining
Myocardial tissue slices were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Sections (5 μm thick) were stained with hematoxylin-eosin (HE) using a standard protocol and analyzed by light microscopy.

| Immunohistochemically staining
ICAM-1 (1:1000 dilution, ab206398; Abcam, Cambridge, UK), P-selectin (1:1000 dilution, sc-8419; Santa Cruz), E-selectin (1:1000 dilution, DF6914; Affinity Biosciences, OH) antibodies were used in F I G U R E 1 After 30 min reperfusion, inject corresponding antibodies 2 mg/kg in femoral vein. At the end of the whole process of reperfusion, we obtain hearts to measure the range of myocardial infarction (N = 6 samples per group). We also collect blood immediately through the femoral vein, then obtain partial myocardial tissue for other experiments (N = 10 samples per group) paraffin embedded heart sections. The results were analyzed by the multi-function color cell image analysis system (Image-Pro Plus V 5.1 software; Media Cybernetics company, Rockville, MD), and system automatically selects 10 meaningful vision, the integral optical density (IOD) was obtained for each view, and 10 views were chosen to calculated the mean IOD.

| Myocardial infarct size measurement
The myocardial infarct size was assessed by Evans Blue (E2129; Sigma, St. Louis, MO) and TTC (T8877; Sigma) double staining method. The viable tissue which was stained red and white by TTC was defined as AAR. The non-ischemic myocardium was stained deep blue by Evans Blue. Infarct area (INF) appeared pale after staining. A percent of infarcted area over total area at risk (INF/AAR ratio, IAR, %) was calculated. 22

| Western Blot analysis
Myocardial tissue (5 mg) was selected to extract the protein for determination of the protein concentration by bicinchoninic acid. The following primary antibodies were used: 1:300 diluted Rabbit anti HMGB1 (DF3077; Affinity Biosciences), TLR4 (AF7017; Affinity Biosciences) and NF-κB (AF5006; Affinity Biosciences) primary antibody. In the next day, added 1:3000 diluted secondary antibody, used ECL for coloration, scanned and analyzed the results by the gel imaging system, used the gray ratio of each protein to beta actin to compare and analyze.

| RT-PCR analysis
Total RNA was extracted from myocardial tissue using the TRIzol reagent (15596026; Ambion, Cambridge, UK), and RNA content measured using 260/280 UV spectrophotometry. The same volume of RNA solution was used to reverse transcriptase by the RT-PCR kit (FSQ-101; TOYOBO, Kita-ku, Osaka, Japan). The mRNA expression of HMGB1, TLR4 and NF-κB were quantified by SYBR Green (170-8882AP; Bio-Rad, Hercules, CA) two-step, real-time RT-PCR using CFX96 Touch Real-Time PCR Detection System. The expression of each gene was normalized to GAPDH mRNA content and calculated using comparative Ct methods. The primers sequence are shown in Table 1.

| Experimental grouping and processing of the in vitro experiments
mDCs was randomly divided into five groups, each group had three time points of 24, 48 and 72 hours, with three wells in each group.

| Test sample collection
After the experiment was completed, culture supernatant was used to detect the cytokine level. RT-qPCR and Western Blotting were performed to detect the expression of MyD88, NF-κB gene and protein in DCs; and flow cytometry was performed to detect the expression of costimulatory molecules.

| Expression of mDCs costimulatory molecules
The detection method was the same as detecting the expression of pbDCs costimulatory molecules in vivo.

| Western Blot analysis
The detection method of MyD88 and NF-κB protein was the same as in vivo. The primary antibodies of Western Blot Analysis were MyD88 (sc-74532; Santa Cruz) and NF-κB (AF5006; Affinity Biosciences).

| RT-qPCR analysis
The detection method of MyD88 and NF-κB mRNA was the same as in vivo by RT-qPCR Analysis. The primers sequence of MyD88, NF-κB are shown in Table 1.

| Co-culture of mDCs and cardiac myocytes
The experiment was divided into the normal oxygen and the hypoxia/ reoxygenation group, each of which was divided into five subgroups. The corresponding drugs were added according to the requirements of the in vitro experimental grouping and treatment. After that, the culture supernatant was taken for cTnI detection. The cardiac myocytes (CMs) in the lower chamber were collected, and the apoptosis was detected by flow cytometry. 22 See the Supplementary Materials and methods for details.

| Statistical analysis
Data were expressed as mean ± SD. Comparisons between groups were assessed by One-way ANOVA followed by Bonferroni's post hoc test. All statistical analyses were performed using Graph Pad Prism Software (Version X, La Jolla, CA). The significance level was set at P < 0.05.

| HMGB1-TLR4 signalling pathway mediates the migration,adhesion and activation of DCs in IR myocardium
After MI/RI, there are great number of inflammatory cells in the myocardium, which was confirmed in HE staining pictures ( Figure 2D) and early studies. 24 In rat IR myocardium, Wang 25 also showed that neutrophil aggregation could be detected after myocardial myeloperoxidase staining. In our rat model, DCs specific marker CD1a and CD80

| HMGB1 antibody attenuates myocardial injury through mediating the role of DCs regulated by the HMGB1-TLR4 signalling pathway
The effects of anti HMGB1 treatment on myocardial damage are illustrated in Figure 3. In our rat model, we have found that: the in-

| Regulation of HMGB1-TLR4 signalling pathway on DCs mediates the migration, adhesion and in ischaemia-reperfusion myocardium
The activation of DCs Regulated by HMGB1-TLR4 signalling pathway is illustrated in Figure 5. In this study, we also found that on the mDCs surface can be analyzed to identify the maturation and activation of DCs. 29 Comparison of expression of CD80 and CD86 in pbDCs (In vivo) and mDCs (In vitro) by flow cytometry see Figure   S3, S4.

| Co-culture of DCs and CMs
The culture, purification and identification of DCs and CMs see Figure S1, S2. Flow cytometry picture of CMs apoptosis rate in coculture see Figure S5.
Comparison of CMs apoptosis rate and cTnI concentration in the co-culture of DCs and CMs are illustrated in Figure 6. In the normal oxygen and hypoxia/reoxygenation group, it was remarkable that HMGB1 stimulation produced a statistically significant decrease

| D ISCUSS I ON
As is known to all, inflammatory response plays an important role in IRI and it is mainly mediated by various inflammatory cells, while its potential cellular and molecular immune mechanisms are quite complex and remains unclear. Apart from macrophages, neutrophils, and lymphocytes, recently, more and more attention has been paid to the role of DCs in IRI. 30 Damage-associated molecular pattern refers to cells that have changed their own conditions (such as under stress or injury) to produce recognizable danger signals, including HMGB1, defensin, etc. 38 HMGB1 is a highly conserved DNA binding protein, which has many extracellular effects besides classical nuclear functions. There are two ways to release HMGB: one is the "active" secretion of the immune system of the body, and the other is the "passive" release of the injured, dead and apoptotic cells. 39 The release of extracellular HMGB1 can play the role of triggering and modulating inflammation. 40   at later time points, macrophages are required for tubular proliferation during normal repair. 46 The effect of DCs is related to its maturity, activation degree and quantity. 28 Dendritic cells not only participates in the inflammatory response after ischaemia, but also affects the healing reaction of IRI. 47  also showed the complexity of the mechanism of HMGB1 functions, and some scholars believe that intravenous injection of HMGB1 before IRI has organ protection effect. 51,52 What's interesting is that F I G U R E 3 High-mobility group protein box-1 (HMGB1) antibody attenuates myocardial injury through mediating the role of dendritic cells (DCs) regulated by the HMGB1-TLR4 signalling pathway. A, Comparison of myocardial infarct size among all groups (n = 6 for each group). Anti-HMGB1 treatment reduces myocardial IR injury. Representative images of the infract area (INF: white), area at risk (AAR: red and white), and normal area (blue). B, Quantitative analysis of infarct size and the INF/AAR ratio (IAR, %). Comparison of concentrations of serum CK-MB, cTnI, HMGB1, and IL-6, IL-8, TNF-α, IL-12 among all groups (n = 10 for each group). Blood was collected right after the IR procedure was completed. CK-MB, cTnI, HMGB1, and IL-6, IL-8, TNF-α, IL-12 were measured as described in given the potential for ischemic heart, after reperfusion, to attenuate the damage caused by currently inevitable IRI that occurs and contributes to the increased risks of following sequelea. In conclusion, HMGB1 is a mediator of heart damage after MI/RI that operates through the TLR4 pathway to activate DCs. Administration of a neutralizing antibody to HMGB1 soon after MI/RI affords significant cardiac protection, which indicates therapeutic potential of this strategy.
In this research, a new function connection was established between the HMGB1/TLR4 pathway and DCs, and it was proved that this signalling pathway can mediate the role of DCs in MI/RI. This experiment fully proves the important role and mechanism of DCs in MI/RI, which makes it possible for the follow-up study of using DCs as the target to protect heart from MI/RI, and make it a promising future.

| CON CLUS ION
Myocardial ischaemia reperfusion injury is very common in clinic as the main pathophysiological process during cardiopulmonary resuscitation, thrombolysis of myocardial infarction and cardiovascular surgery under cardiopulmonary bypass. Therefore, to reduce the occurrence of MI/RI and find safer and more effective treatments has been an important research topic of the cardiovascular field.
Through in vivo and in vitro cell experiments, the following conclusions are reached in this study: In case of MI/RI, the migration, adhesion and aggregation of DCs to myocardium are increased.

E THI C S APPROVAL AND CON S ENT TO PARTI CIPATE
The animal procedures were approved by Wenzhou Medical University Animal Care and Use Committee (No: wydw2014-0058), which were

AVAIL AB ILIT Y OF DATA AND MATERIAL
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

ACK N OWLED G EM ENT
We would like to thank "iCell Bioscience Inc" in shanghai for the help with isolation, culture, purification and identification of rat DCs, and Dr. Chunxiang Zhang of Rush University Medical Center in Chicago for the help with the manuscript preparation.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R S' CO NTR I B UTI O N S
Qifeng Zhao conceived of the study, and participated in its design and coordination and helped to revised the manuscript. Jiyang