Tolerogenic dendritic cells induced by atorvastatin via inhibition of the TLR-4/NF-κB pathway improve cardiac remodeling after myocardial infarction

Background Necrosis of ischemic cardiomyocytes after myocardial infarction (MI) activates an intense inammatory reaction. Dendritic cells (DCs) play a crucial role in the repair process after MI. Tolerogenic DCs (tDCs) can inhibit inammatory responses. Methods and results We investigated the role of atorvastatin and supernatants of necrotic cardiomyocytes (SNC) on DCs. We found that SNC induced DCs maturation, activated TLR-4/NF-κB pathway, promoted inammatory cytokines secretion and oxidative stress. Co-treatment with SNC and atorvastatin suppressed DC maturation and inammatory response, which meant that atorvastatin induced DCs tolerate to SNC. Then, we investigated the effect of mDCs induced by SNC and tDCs induced by atorvastatin on ventricular remodeling after MI. tDCs treatment signicantly improved the left ventricular systolic function, reduced the inltration of MPO + neutrophil, Mac3 + macrophages and CD3 + T cells, inhibited myocardial apoptosis and brosis, and decreased infarct size. Compared with PBS, treatment with mDCs did not showed benecial effect on ventricular remodeling and inammatory reaction after MI in mice. Atorvastatin the TLR-4/NF-κB repressed the oxidative stress, inammatory response, and immune maturity induced by SNC. Treatment with tDCs, induced by co-treated with atorvastatin, preserved left ventricular function, limited infarct size, suppressed the inltration of inammatory cells, and attenuated the severity of brosis, and reduced the of cardiomyocytes. effects, including improved endothelial dysfunction, increased nitric oxide bioavailability and antioxidant properties, inhibition of inammatory processes, and stabilization of atherosclerotic plaques [19]. In a previous study, we demonstrated that atorvastatin inhibited AngII-induced inammatory responses via the downregulation of DC surface markers and inammatory cytokines [11]. In this study, we showed that atorvastatin inactivated the TLR-4/NF-κB pathway, repressed the inammatory response, and immune maturity induced by SNCs.


Introduction
Myocardial infarction (MI) remains the leading cause of death worldwide [1]. Necrosis of ischemic cardiomyocytes in the infarcted myocardium activates both myocardial and systemic in ammatory responses. For example, animal experiments have suggested that in ammatory cells in ltrating the infarcted myocardium may exacerbate an ischemic injury, causing the death of viable cardiomyocytes and adverse left ventricular remodeling. However, clinical trials examining anti-in ammatory approaches in patients with myocardial infarction did not improve outcomes and failed to reduce the size of the infarct [2].
In recent decades, new efforts have pursued therapeutic strategies aimed at exploiting stem cell biology in the preservation, regeneration, or repair of cardiac tissue post-MI. However, a recent meta-analysis assessing the results of stem cell therapy for patients with acute MI demonstrated no net bene cial effects on outcomes, except for a small improvement in the ejection fraction. Although some stem cells differentiate to express markers characteristic of cardiomyocytes, the majority of studies demonstrated that the potential bene t derived from stem cells does not result from trans-differentiating into the target tissue but from their paracrine activities, such as secretion of several cytokines and growth factors related to the inhibition of in ammation and adverse left ventricular remodeling [3]. Thus, we put forward the hypothesis that the adoptive transfer of a specialized regulatory immune cell after MI may have better outcomes than stem cell therapy.
Dendritic cells (DCs) are the primary professional antigen-presenting cells, uniquely able to induce naïve T cell activation and effector differentiation. Accumulating evidence strongly suggests that DCs are much more versatile in their functions than previously thought [4]. For example, DCs have been identi ed as relevant sources of cytokines in the healing infarct. Additionally, our previous studies have suggested that the immune maturation of DCs may play a crucial role in atherosclerotic lesions and the repair process after MI [5][6][7][8][9][10]. We found that the supernatants of necrotic cardiomyocytes (SNCs) induced the maturation and in ammatory reaction of DCs [9][10]. Our previous study also demonstrated that atorvastatin inhibited Angiotensin II (AngII)-induced in ammatory responses via the downregulation of DC surface markers and in ammatory cytokines [11]. Thus, the goal of this study is to investigate the role of atorvastatin on DC maturation induced by SNCs, and the effect of treatment with DCs treated with atorvastatin after MI.

Materials And Methods
Simulation of post-MI cardiomyocyte microenvironment Two types of samples were used to mimic the MI microenvironment: 1. the supernatants of necrotic HL-1 cells (Supernatant-NH), 2. the supernatants of infarcted myocardium (Supernatant-IM) at 8 h post-MI. The generation of necrotic HL-1 cells was performed according to the protocol described by Maekawa et al. [12]. Brie y, HL-1 (a cardiac muscle cell line; SXBIO, Shanghai, China) cells were washed three times with serum-free medium and then processed by ve cycles of freezing in liquid nitrogen followed by thawing at 37 °C. Supernatants from infarcted mouse myocardium were obtained from MI-model mice after ligation of the left anterior descending (LAD) artery for 8 h. In brief, the hearts of the mice were removed under sterile conditions, and sections of infarcted myocardium were excised and washed three times in phosphate-buffered saline (PBS) to remove blood cells. Following this, the sections were cut into small pieces that were used to generate single-cell suspensions via the gentleMACS™ system (MACS® Cell Separation, Miltenyi Biotec B.V.& Co., Bergisch Gladbach, Germany). Finally, the supernatants were obtained by centrifugation. Then, 100-µL samples (cell membrane particles were removed by centrifugation at 1500 g for 30 min) were collected from the supernatants of the HL-1 cells, and infarcted myocardial cells were added to 10 6 bone marrow-derived dendritic cells (BMDCs) for 24 h. All experiments were performed under sterile conditions. All procedures performed in studies involving animals were in accordance with the ethical standards of Laboratory Animal Management and experimental ethics committee of Union Hospital, Fujian Medical University. All methods were performed in accordance with relevant guidelines and regulations.

Cell culture and treatments
The BMDCs obtained from approximately 6-week-old C57BL/6 mice were cultured in RPMI 1640 Media supplemented with 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1 ng/mL IL-4 at 37 ∘ C in 5% humidi ed CO 2 for 4 h. The medium containing nonadherent cells was replaced with fresh medium every 2 d. On culture day 7, the cells were treated with Supernatant-NH and Supernatant-IM alone, or in combination with 10 M atorvastatin (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. PBS was used as a control.

Induction of MI models and injection of DCs
Ligation of the left main descending coronary artery (LCA) was performed as described previously [9]. In brief, mice were anesthetized with 2% iso urane, and their hearts were manually exposed through small
Immunoreactive proteins were identi ed using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scienti c, Franklin, MA, USA). Densitometric analysis of the western blotting was performed using Image J software.

Echocardiography
In vivo cardiac function was determined by echocardiography using the Vevo ® 2100 Imaging Platform (VisualSonics, Inc., Toronto, Canada) as described previously [9]. Brie y, the mice were anesthetized with 2% iso urane and oxygen, and two-dimensional echocardiographic views of the left ventricular long axis through the anterior and posterior LV walls were obtained at the level of the papillary muscle tips below the mitral valve. The left ventricular ejection fraction (LVEF) and fractional shortening (FS) were calculated as previously reported [6]. The echocardiograms were evaluated in a blinded manner.

Measurement of myocardial infarct size and myocardial brosis
After freezing at −20 °C for 12 hours, the heart ventricles were sectioned transversely into ~2-mm thick sections. The sections were subsequently incubated in 1% triphenyltetrazolium chloride (TTC) for 15 min at 37 °C to identify the non-infarcted and infarcted areas. Once identi ed, the areas were xed in 10% buffered formalin. The infarcted area was displayed as the TTC-unstained area (white). The extent of brosis was measured using Masson's trichrome stain on day 28 after MI. Image-Pro Plus software (v6.0; Media Cybernetics, Rockville, MD, USA) was used to determine the infarct size and extent of brosis.

Immunohistochemistry analysis
Immunohistochemical studies were performed by immunoperoxidase staining methods using para nembedded tissue sections (6 mm thick). After inhibiting endogenous peroxidase activity, the sections were incubated with primary anti-myeloperoxidase (MPO; Abbiotec™, Midlothian, UK), anti-Mac3 (BD Biosciences, San Jose, CA, USA), and anti-CD3 (eBioscience, Inc., San Diego, CA, USA) at 4 °C overnight, followed by respective secondary HRP-conjugated antibodies for 1 h at room temperature. The positive cells were visualized with DAB, and nuclei were counterstained with hematoxylin. The numbers of Mac3+ macrophages, MPO+ neutrophils, and CD3+ T lymphocytes were assessed by counting the total cell numbers in the infarcted and border areas in twenty randomly chosen elds in each section.

Cardiomyocyte apoptosis
Cardiomyocyte apoptosis was assessed in the heart sections by terminal deoxynucleotidyl transferasemediated dUTP nick-end-labeling (TUNEL) staining. TUNEL staining was performed using the In situ Cell Death Detection Kit (Roche Applied Science, Upper Bavaria, Germany) according to the manufacturer's protocol. The apoptosis index was determined by counting TUNEL-positive nuclei in ten random elds per section and expressed as a percentage of the total nuclei.

Statistical analyses
The data are presented as the means ± SD, with < 0.05 considered to be statistically signi cant. A oneway ANOVA, followed by the Student-Newman-Keuls (SNK) test, was employed for the statistical analysis of our results. All statistical analyses were performed with SPSS 21 statistical software.

Effect of atorvastatin on DCs maturation and in ammatory cytokine secretion
There were several observable costimulatory proteins on the surface of BMDCs, including CD40, CD80, and CD86. These proteins were examined in DCs that were treated with Supernatant-NH and Supernatant-IM alone, or in combination with 10 M atorvastatin. The ow cytometry results showed that Supernatant-NH and Supernatant-IM upregulated the expression of the cell-surface markers CD40, CD80, and CD86. We subsequently observed that atorvastatin signi cantly downregulated CD40, CD80, and CD86. These results indicate that atorvastatin may suppress DC maturation induced by Supernatant-NH and Supernatant-IM (Fig. 1).
An analysis of cytokine levels, that is, TNF-, IL-1, IL-6, IL-12P40, and IL-8, indicated an in ammatory response, which was induced by Supernatant-NH and Supernatant-IM in DCs. We found that the secretion of TNF-, IL-1, IL-6, IL-12P40, and IL-8 was increased signi cantly in the presence of Supernatant-NH and Supernatant-IM. In addition, the secretion of these cytokines was suppressed by atorvastatin ( Fig. 1).

Atorvastatin attenuates oxidative stress in DCs
To investigate how atorvastatin affects the oxidative stress in DCs induced by Supernatant-NH and Supernatant-IM, we used ow cytometry to measure the intracellular level of ROS in response to Supernatant-NH and Supernatant-IM stimulation. Moreover, we also measured the levels of MDA and SOD with ELISA kits. As depicted in Figure 2, Supernatant-NH and Supernatant-IM exhibited a stimulating effect on ROS and MDA production but inhibited SOD activity. Cotreatment with atorvastatin reduced intracellular ROS production, and similar results were observed with MDA. However, atorvastatin suppressed the inhibition of SOD activity induced by Supernatant-NH and Supernatant-IM. Therefore, these results suggest that atorvastatin signi cantly suppresses oxidative stress in DCs induced by Supernatant-NH and Supernatant-IM.

Effect of DCs transfer on the survival and LV function in MI mice
As demonstrated above, Supernatant-IM induced DC maturation, activated in ammatory signal pathways, promoted in ammatory cytokine secretion, and oxidative stress. Nonetheless, co-treatment with atorvastatin suppressed DC maturation and in ammatory cytokine secretion, inactivated the TLR-4/NF-κB pathway, suggesting that atorvastatin induced DCs to tolerate Supernatant-IM. Therefore, we investigated the effect of mDCs induced by Supernatant-IM, tolerogenic DCs (tDCs) induced by Supernatant-IM, and atorvastatin co-treatment on the survival and LV function in MI mice.
Compared to PBS and mDCs treatment after MI, tDCs treatment signi cantly improved the LVEF at 4 weeks after MI. Both the LVEDD and LVESD were reduced in tDCs treatment mice. The survival rate trended towards being higher in the tDCs-treated mice compared with the PBS-and mDCs-treated mice on day 28. No bene cial effects on cardiac function were observed in the mDCs group. These ndings demonstrated that tDCs treatment might attenuate adverse remodeling and improve LV systolic function ( Figure 4).

Effect of DCs transfer on the in ammatory reaction after MI
Immunohistochemical staining for MPO showed that the number of neutrophils that in ltrated into the infarcted myocardium was signi cantly reduced in the tDCs group at 3 and 7 d after MI than in the PBS and mDCs group. Similarly, tDCs treatment signi cantly decreased Mac3+ macrophage in ltration compared to the PBS and mDCs group. The number of CD3+ T cells that in ltrated the infarcted myocardium was also downregulated in the tDCs group. Compared with the PBS group, mDCs treatment did not reduce the in ltration of in ammatory cells into the infarcted heart ( Figure 5).

Effect of DCs transfer on infarct size, apoptosis, and brosis in MI mice
The number of myocardial apoptotic nuclei, as detected by TUNEL staining, was signi cantly lower in the LV border zone of tDCs treatment mice compared with PBS and mDCs treatment mice on day 3 after MI. TTC staining on the rst day after MI demonstrated a signi cantly smaller infarct size in the tDCs group. On day 28, Masson's trichrome staining showed that tDCs treatment signi cantly reduced LV brosis. On the contrary, mDCs treatment did not show a reduction in apoptosis, infarct size, or brosis. (Figure 6).

Discussion
The initial nding of our study is that atorvastatin inactivated the TLR-4/NF-κB pathway as well as repressed the in ammatory response, oxidative stress, and immune maturity induced by SNCs. Secondly, treatment with tDCs, which were induced by co-treatment with atorvastatin and SNCs, preserved left ventricular function, limited the infarct size, suppressed the in ltration of in ammatory cells, attenuated the severity of brosis, and reduced the number of apoptotic cardiomyocytes.
DCs are antigen-presenting cells that are highly involved in the process of myocardial infarction [13]. Ferrans et al. demonstrated the rapid accumulation of interstitial DCs in the border zones 7 d after myocardial infarction (left coronary artery ligation) in the rat heart. DCs tend to be assembled in small clusters with CD4 + T cells, which disappear 21 d after coronary ligation [14]. Anzai and colleagues reported that selective depletion of DCs exacerbated post-infarction LV remodeling in association with enhanced in ammatory cytokine expression, inducible nitric oxide synthase production, and MMP-9 activation [15]. Furthermore, the cardioprotective effects of ACEI were enhanced through attenuating migration of DCs from the spleen into peripheral circulation, thereby inhibiting DCs maturation and tissue in ammation [16]. Toll-like receptors (TLRs) are a family of pattern recognition receptors expressed on DCs. Numerous studies have demonstrated that TLR4 activates the expression of several proin ammatory cytokine genes and induces immune maturity in DCs [17]. Recent studies have supported the notion that TLR4 of the bone marrow-derived cells plays an essential role in mediating cardiac dysfunction under certain pathological conditions [18].
In our previous studies, we used SNCs to simulate the post-MI cardiomyocyte micro-environment in vitro. We demonstrated that necrotic supernatants up-regulated the expression of DC maturation markers, and increased the levels of in ammatory cytokines [9,10]. In the present study, we found that SNCs activated the TLR-4/NF-κB pathway as well as induced the in ammatory response, oxidative stress, and immune maturity.
Statins, the cornerstone of antiatherogenic therapy, possess pleiotropic effects, including improved endothelial dysfunction, increased nitric oxide bioavailability and antioxidant properties, inhibition of in ammatory processes, and stabilization of atherosclerotic plaques [19]. In a previous study, we demonstrated that atorvastatin inhibited AngII-induced in ammatory responses via the downregulation of DC surface markers and in ammatory cytokines [11]. In this study, we showed that atorvastatin inactivated the TLR-4/NF-κB pathway, repressed the in ammatory response, and immune maturity induced by SNCs.
DCs exist in an immature and mature state. Immature or semi-mature DCs can induce tolerance, also known as tDCs. Several studies have investigated a number of methodologies for inducing tDCs and the treatment effect on a variety of diseases [20]. It has been documented that tDCs, generated by using vitamin D 3 , showed a stable maturation-resistant semi-mature phenotype with a low expression of activating co-stimulatory molecules as well as no production of the IL-12 family of cytokines. Furthermore, several studies demonstrated that tDCs induced by vitamin D 3 decreased islet rejection in pancreatic islet transplantation and prolonged male skin grafts in female recipients [21]. Vitamin D 3 , dexamethasone, and rapamycin are also used to induce tolerogenic DCs, which are being further investigated in animal experiments for organ transplantation rejection and certain in ammatory diseases [20].
Recently, several studies have also reported on the results of tDCs treatment for MI. Zhu and colleagues demonstrated that the adoptive transfer of IL-37 plus troponin-treated tDCs attenuated the in ltration of in ammatory cells in infarct hearts, decreased myocardial brosis, and improved cardiac function [22]. Choo and colleagues showed that subcutaneously administered tDCs resulted in better wound remodeling, preserved left ventricular systolic function after myocardial tissue damage, and improved survival [23]. In the present study, we found that atorvastatin inhibited the in ammatory response and immune maturity of DCs treated with SNCs. Therefore, we investigated the treatment effect of tDCs induced by atorvastatin and SNCs in MI mice, and found that tDCs preserved left ventricular function, limited infarct size, suppressed the in ltration of in ammatory cells, attenuated the severity of brosis, and reduced the number of apoptotic cardiomyocytes.

Conclusions
The present study demonstrated that atorvastatin inactivated the TLR-4/NF-κB pathway, inhibited the immune maturity induced by SNCs. Furthermore, the adoptive transfer of tDCs induced by co-culturing with atorvastatin and SNCs preserved left ventricular function, limited infarct size, suppressed the in ltration of in ammatory cells, attenuated the severity of brosis, and reduced the number of apoptotic cardiomyocytes.

Declarations
Ethics approval and consent to participate The study design was approved by the ethics committee of Union Hospital, Fujian Medical University. All procedures performed in studies involving animals were in accordance with the ethical standards of Laboratory Animal Management and experimental ethics committee of Union Hospital, Fujian Medical University. All methods were performed in accordance with relevant guidelines and regulations.
Consent for publication All the authors have approved the manuscript and have agreed to submission to your esteemed journal. This manuscript has not been published or presented elsewhere in part or in its entirety, and is not under consideration by another journal.
Availability of data and materials All data generated or analysed during this study are included in this published article.
Competing interests The authors declare that there are no con icts of interest.  The SOD activity and (c) MDA levels were measured at 450 and 532 nm, respectively, using ELISA kits.