Dectin-1 Contributes to Myocardial Ischemia/Reperfusion Injury by Regulating Macrophage Polarization and Neutrophil Infiltration

Introduction

The rapid restoration of blood flow through the occluded coronary artery by primary percutaneous coronary intervention constitutes the most effective therapy for limiting the infarct size and improving the clinical outcome after acute myocardial infarction (MI).¹ Compared with thrombolytic therapy, primary percutaneous coronary intervention results in reduced mortality and morbidity; however, ischemia/reperfusion (IR) injury remains an important complication, contributing to up to 50% of the final infarct size.¹ IR injury is a multifactorial process including the activity of metabolic factors, inflammation, oxidative stress, and microvascular obstruction.^1,2 To date, no treatments targeting this process have shown conclusive benefits.

Accumulating evidence, including ours, has demonstrated that the immune response plays a central role during IR injury and that this response is characterized by the recruitment and activation of immune cells associated with the innate and adaptive immune system.^2–4 Pattern recognition receptors (PRRs) such as Toll-like receptors, NOD-like receptors, C-type lectin receptors, and RIG-1–like receptors are expressed by the innate inflammatory cells.⁵ These receptors can be activated by the IR injury, leading to the activation of signaling mediators, which is followed by the transcriptionally regulated production of proinflammatory mediators, including cytokines, chemokines, and adhesion molecules, thus leading to tissue inflammation and, in turn, affecting IR injury.² Accordingly, therapeutic targeting of these innate receptors, signaling molecules, and inflammatory mediators may attenuate the IR injury.

Dectin-1 is a member of the C-type lectin receptor family, and it is expressed mainly on activated myeloid cells such as macrophages, dendritic cells, or neutrophils.⁶ Dectin-1 signaling is involved in the regulation of numerous cellular responses, including phagocytosis, autophagy, respiratory burst, and the production of inflammatory lipids and numerous cytokines and chemokines, including Th17-polarizing cytokines such as interleukin (IL)–23, IL-6, and IL-1β.^7,8 Dectin-1 plays crucial functions in antifungal immunity, and similar to many other PRRs, it has been reported that Dectin-1 may be involved in the regulatory processes in immune homeostasis, autoimmunity, allergy, and cancer.^6,7 Therefore, Dectin-1 may represent a therapeutic target in the treatment of both infectious and noninfectious diseases. In addition to Dectin-1, our recent study demonstrated that Dectin-2 leads to an increase in cardiac rupture, impairs wound healing, and aggravates cardiac remodeling after MI through the modulation of Th1 differentiation.⁹ However, whether Dectin-1 contributes to myocardial IR injury and the potential underlying mechanisms is largely unknown.

In this study, we investigated the expression of Dectin-1 in ischemic and reperfused myocardium and characterized the functional involvement of Dectin-1 in myocardial IR injury. We show that Dectin-1 is expressed largely on cardiac macrophages but not on neutrophils and that it plays a pathogenic role in myocardial IR injury by inducing proinflammatory M1 macrophage polarization and Ly-6C⁺ monocyte and neutrophil infiltration.

Methods

The data, analytical methods, and study materials are available from the corresponding author on reasonable request.

An expanded Methods section is available in the online-only Data Supplement.

Mice

Dectin-1^−/− (D1KO; gene symbol, Clec7a) mice on a C57BL/6 background were generated as described previously.¹⁰ Wild-type (WT) C57BL6/J mice (weight, 20–25 g; male; 10–16 weeks old; SLRC Laboratory Animal, Shanghai, China) were used as controls in this study. This study and all animal procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication No. 85-23, revised 1996) and were approved by the Animal Care Committee of Shanghai Jiaotong University School of Medicine.

In Vivo Myocardial IR Protocol

Surgical induction of myocardial IR was performed as previously described.⁴ Briefly, mice were lightly anesthetized with diethyl ether, intubated, and fully anesthetized with 1.0% to 1.5% isoflurane gas, and mechanical ventilation was performed with a rodent respirator. A left thoracotomy was performed, and the left anterior descending coronary artery was visualized with a microscope and ligated at the site of its emergence from the left atrium with 8-0 silk suture around fine PE-10 tubing with a slipknot. Complete occlusion of the vessel was confirmed by the presence of myocardial blanching in the perfusion bed. Mice underwent 45-minute-long left anterior descending artery ischemia followed by different periods of reperfusion. Sham-operated animals were subjected to the same surgical procedures except that the suture was passed under the left anterior descending artery but not tied.

Additional Methods

The expanded Methods section in the online-only Data Supplement contains information on the treatment and groups; data on clinical study participants; a description of flow cytometric analyses; quantification of absolute cell numbers; quantitative real-time polymerase chain reaction; immunohistochemistry, echocardiographic, and hemodynamic analyses of cardiac function; infarct size assessment; Western blotting; myocardial apoptosis; bone marrow transplantation (BMT); and cell culture and ELISA. All antibodies and kits used in this study are listed in Table I in the online-only Data Supplement.

Statistical Analysis

All values are presented as mean±SEM or median with interquartile ranges as appropriate. Comparisons between 2 groups were made with the Mann-Whitney U test, whereas data obtained from multiple groups were compared with the Kruskal-Wallis test with the Dunn multiple comparison test or Bonferroni post hoc analysis. Two-way ANOVA followed by Bonferroni post hoc analysis was performed to analyze data with 2 factors. The Spearman rank correlation analysis was used to test the relationship between variables. Values of P<0.05 were considered statistically significant. Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Prism Software Inc, San Diego, CA) and SPSS 15.0 for Windows (SPSS, Inc, Chicago, IL).

Results

Cardiac Dectin-1 Expression Increased After Myocardial IR Injury

To elucidate the involvement of Dectin-1 in the myocardial IR injury, we first investigated Dectin-1 expression levels in the myocardium at different reperfusion time points after the IR. Dectin-1 mRNA and protein expression levels were shown to increase significantly as early as 12 hours after the reperfusion and to rise progressively until 24 hours. Afterward, they reached their peak values and began to decrease, approaching the baseline levels at 72 hours after reperfusion (Figure 1A and 1B). Moreover, Dectin-1 expression was significantly higher in the infarct area compared with the noninfarct area in the WT C57BL/6 mice, whereas no Dectin-1 expression was observed in the D1KO mice (Figure 1A and 1B). Immunohistochemical analyses of murine models confirmed that Dectin-1 was expressed mainly in the infarct and border areas after myocardial IR injury, with low expression in the noninfarct areas (Figure 1C).

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Next, we attempted to identify the leukocytes affecting Dectin-1 expression after myocardial IR injury. Flow cytometric analysis revealed that almost all Dectin-1–expressing cells were CD11b⁺ myeloid cells in the hearts at day 1 after IR; however, the lymphocytes did not express Dectin-1 (Figure 1D and Figure I in the online-only Data Supplement). Among CD11b⁺ myeloid cells, nearly 80% of CD11b⁺F4/80⁺Ly-6G⁻ macrophages expressed Dectin-1, whereas CD11b⁺Ly-6G⁺F4/80⁻ neutrophils made up only a minor portion (≈2.2%) of Dectin-1–expressing leukocytes (Figure 1E and Figure I in the online-only Data Supplement). Double-immunofluorescence staining of Dectin-1 together with F4/80 or Ly-6G staining confirmed that F4/80⁺ macrophages, but not Ly-6G⁺ neutrophils, expressed mainly Dectin-1 as well in the murine hearts at day 1 after IR (Figure 1F).

Dectin-1 Deficiency Ameliorated Myocardial IR Injury

To elucidate the impact of Dectin-1 loss on myocardial IR injury, we compared the severity of myocardial IR injury between D1KO and WT C57BL/6 mice. Before the surgery, we excluded the possibility of any cardiac abnormalities in D1KO mice by performing echocardiography. Echocardiographic parameters of D1KO mice were comparable to those of the WT mice (Table II in the online-only Data Supplement). Initially, we confirmed the absence of Dectin-1⁺ cells in D1KO mice by flow cytometry (Figure 2A and Figure II in the online-only Data Supplement). D1KO and WT mice were then subjected to the myocardial IR injury. Dectin-1 expression levels peaked at day 1 after IR and remained elevated for 2 days; therefore, we assessed the extent of injury and cardiac function. The extent of cardiac ischemic injury was compared at day 2 after the initiation of reperfusion. With similar size of areas at risk (AARs), the infarct area/AAR ratio was shown to be significantly lower in D1KO than in WT mice (26.53±1.52% versus 41.72±2.32%, respectively; Figure 2B and 2C and Figure III in the online-only Data Supplement).

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To determine the effects of Dectin-1 on cardiac function after IR and to demonstrate the clinical relevance of our findings, we measured the left ventricular (LV) end-diastolic volume (EDV), LV end-systolic volume (LVESV), and ejection fraction (EF) by using echocardiography at day 4 after myocardial IR injury. Both LVEDV and LVESV were significantly increased and EF was considerably decreased compared with those measured in the sham-operated group. However, Dectin-1 knockout led to a significant decrease in LVEDV (69.90±1.17 μL versus 84.49±1.51 μL) and LVESV (35.56±0.59 μL versus 49.92±1.03 μL), as well as an increase in EF (50.24±0.60% versus 40.91±0.73%), accompanied by other improvement of LV diameters (LV end-diastolic diameter, LV end-systolic diameter, and fractional shortening), compared with the WT mice after the IR (Figure 2D and 2E and Table III in the online-only Data Supplement), indicating an improved cardiac function.

After this, to test whether Dectin-1 affects hemodynamic properties, we measured LV end-diastolic pressure (LVEDP), LV end-systolic pressure, mean arterial pressure, and the derivative of LV pressure (dP/dt) using a Millar catheter. The LV hemodynamic status was augmented in D1KO mice compared with the WT mice at day 4 after IR. Specifically, LVEDP was lower (4.6±0.8 mm Hg versus 8.1±1.2 mm Hg, respectively), whereas the indexes of contractility, the maximal rate of the increase of left ventricular pressure (dP/dtmax) (4810±241 mm Hg/s versus 3813±212 mm Hg/s, respectively) and maximal rate of the decrease of left ventricular pressure (dP/dtmin) (−4652±178 mm Hg/s versus −3591±189 mm Hg/s, respectively), were significantly increased in the D1KO mice compared with the WT mice (Table III in the online-only Data Supplement), suggesting an improved cardiac function in D1KO mice after IR.

Ischemia-induced cardiomyocyte apoptosis has been demonstrated to be critically involved in myocardial IR injury.¹¹ On the basis of these findings, we performed terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphate nick-end labeling of LV sections obtained from WT and D1KO mice at 24 hours after myocardial IR. As shown in Figures 2F and 2G and Figure IV in the online-only Data Supplement, IR stress led to an increase in the rate of cardiomyocyte apoptosis compared with the sham-operated group, and the number of terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphate nick-end labeling–positive cardiomyocytes was significantly decreased in the DK1O mice after IR compared with the WT mice. Notably, the difference was substantially more significant in the infarct area and the border area. In line with these findings, caspase-3 activity, analyzed with a caspase colorimetric assay, in the ischemic myocardium was concomitantly downregulated in the D1KO mice (Figure 2H). Moreover, the protein levels of the proapoptosis molecules Bax and cleaved caspase-3 decreased and the antiapoptosis molecule Bcl-2 increased in the LV of D1KO mice compared with the WT mice after the IR (Figure 2I and 2J).

Furthermore, Dectin-1 and Dectin-2 double-knockout (D1D2 DKO) mice were generated to further examine the function of Dectin-1 beyond that of Dectin-2 (Figure VA in the online-only Data Supplement). The extent of injury and cardiac function were assessed and compared among the WT, D1KO, Dectin-2 knockout, and D1D2 DKO mice. The infarct area/AAR ratio at day 2 after IR was shown to be significantly lower in D1D2 DKO mice compared with the other groups (23.71±2.38% [D1D2 DKO] versus 27.45±2.79% [D1KO], 30.88±2.50% [Dectin-2 knockout], and 45.50±2.65% [WT], respectively; Figure VB and VC in the online-only Data Supplement). In addition, cardiac function was significantly improved in D1D2 DKO mice at day 4 after IR injury, as indicated by decreased LVEDV and LVESV, along with increased EF (55.82±0.71% [D1D2 DKO] versus 49.83±0.43% [D1KO], 44.16±0.52% [Dectin-2 knockout], and 39.45±0.74% [WT]; Figure VD and VE in the online-only Data Supplement), indicating that Dectin-1 and Dectin-2 are not redundant in myocardial IR injury.

Dectin-1 Expressed on Bone Marrow–Derived Cells Was Shown to Mediate Myocardial IR Injury

To further investigate the importance of Dectin-1 expression on the infiltrated inflammatory cells in myocardial IR injury, we conducted BMTs between D1KO and WT mice. Because D1KO mice had a C57B/6 background expressing CD45.2, we used a substrain of C57B/6 mice expressing the CD45.1 allele (C57B/6-CD45.1), the same as the WT mice, to facilitate the monitoring (Figure 3A–3C). Flow cytometric analyses confirmed that the WT C57B/6-CD45.1 (CD45.1-WT) mice express the CD45.1 allele, whereas D1KO mice express CD45.2 (Figure 3A). Eight weeks after the BMTs, we demonstrated that the bone marrow of the recipient mice was successfully reconstituted with the donor bone marrow cells by analyzing peripheral blood cells with flow cytometry (Figure 3B). Genotyping analyses also demonstrated that the reconstitution was equally efficient in all transplant groups (Figure VI in the online-only Data Supplement). Recipient mice that underwent successful BMT were subjected to IR, and we further showed that the bone marrow–derived donor cells infiltrated into the heart and expressed Dectin-1 at day 1 after IR (Figure 3C).

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The extent of injury was assessed at day 2, and heart functions were analyzed at day 4 after IR in recipient mice. With similar AARs, the transplantation of D1KO bone marrow–derived cells to WT mice (D1KO BM→WT) led to a significant decrease in the ratio of the infarct area to AAR compared with the WT mice transplanted with WT bone marrow (WT BM→WT; 28.01±1.91% versus 44.10±2.26%, respectively). In contrast, transplantation of WT bone marrow–derived cells to D1KO mice (WT BM→D1KO) led to a considerable increase in the infarct size compared with the D1KO mice transplanted with D1KO bone marrow–derived cells (D1KO BM→D1KO) (45.40±2.16% versus 27.10±1.58%, respectively; Figure 3D and 3E).

Consistent with these findings, we observed improved cardiac function as indicated by higher EF (49.91±0.40% versus 40.13±0.75%), lower LVEDP (4.9±0.7 mm Hg versus 8.3±0.7 mm Hg), and higher LV dP/dt (dP/dtmax 4673±175 mm Hg/s versus 3561±221 mm Hg/s; dP/dtmin −4529±226 mm Hg/s versus −3681±176 mm Hg/s) in the D1KO BM→WT group after myocardial IR compared with the WT BM→WT mice, respectively. However, cardiac function characterized by these parameters was significantly aggravated in D1KO mice transplanted with the WT bone marrow (Figure 3F and 3G and Table IV in the online-only Data Supplement), implying that the extent of injury and cardiac function are determined by the Dectin-1 genotype of the bone marrow cells. These results clearly indicated that Dectin-1 expressed on bone marrow–derived inflammatory cells critically contributes to myocardial IR injury.

Dectin-1 Induced Proinflammatory M1 Macrophage Polarization and Neutrophil Infiltration After Myocardial IR

Next, we studied the influx of neutrophils and macrophage polarization in mouse hearts at day 1 after IR by using flow cytometry. In Figure 4A, the gating strategy for neutrophils (CD11b⁺Ly-6G⁺), Ly-6C⁺ monocytes (CD11b⁺Ly-6G⁻Ly-6C⁺), proinflammatory M1 macrophages (CD11b⁺Ly-6G⁻Ly-6C⁻CD64⁺F4/80⁺CD206⁻), and anti-inflammatory M2 macrophages (CD11b⁺Ly-6G⁻Ly-6C⁻CD64⁺F4/80⁺CD206⁺) is presented. The obtained results demonstrated that Dectin-1 deficiency led to a significant reduction in neutrophil and Ly-6C⁺ monocyte infiltration, accompanied by a relative increase in macrophage influx, compared with the WT mice (Figure 4A and 4B). However, among all tissue-infiltrating macrophages, we observed a suppressed infiltration of F4/80⁺CD206⁻ proinflammatory M1 macrophages in the hearts of D1KO mice and the enhanced recruitment of F4/80⁺CD206⁺ anti-inflammatory M2 macrophages compared with the WT mice at day 1 after IR (Figure 4C and 4D). In addition, Dectin-1 deficiency decreased Ly-6C⁺ monocyte recruitment from the circulation to the heart after cardiac IR injury (Figure VII in the online-only Data Supplement).

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In addition, we examined the temporal changes in the expression of M1 (tumor necrosis factor [Tnf-α], IL-1β, inducible nitric oxide synthase [iNOS], IL-23p19) and M2 (mannose receptor [MR], Arg1) signature genes in the heart after IR. In the WT mice, the gene expression of Tnf-α, IL-1β, Nos2, and Il-23p19, which represent proinflammatory cytokines associated with M1 macrophage polarization, increased at day 1 after IR and gradually decreased thereafter, whereas in D1KO mice, the expression of these M1-associated genes at days 1 and 3 after IR was significantly suppressed (Figure 4E). However, the expression of M2-related genes was enhanced in D1KO mice compared with the WT mice after IR (Figure 4F).

Dectin-1 Was Shown to Mediate Neutrophil Recruitment Through the Regulation of Chemokine (C-X-C motif) Ligand 1 and Granulocyte–Colony-Stimulating Factor Expression After IR In Vivo

Neutrophil infiltration is a hallmark of an inflammatory injury after myocardial IR.^2,4 Therefore, we examined the mechanism underlying Dectin-1 effects on neutrophil influx after IR. The expression levels of chemokine (C-X-C motif) ligand 1 (Cxcl1) and granulocyte-colony stimulating factor (G-CSF), well-known neutrophil chemoattractants, rapidly increased after myocardial IR injury, peaked at day 1, and decreased thereafter (Figure 5A). However, Dectin-1 deficiency considerably reduced Cxcl1 and G-CSF expression at both the mRNA and protein levels, especially at day 1 after IR. The expression of Cxcr2 and G-CSF receptor (G-CSFR) also was significantly decreased in D1KO mice after IR because Cxcr2 is the major Cxcl1 receptor, whereas G-CSFR is the major G-CSF receptor (Figure 5B).

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Because macrophages represent one of the main sources of Cxcl1 and G-CSF, we further demonstrated that Cxcl1 and G-CSF levels were significantly downregulated in the sorted macrophages obtained from the IR hearts of D1KO mice compared with those in the macrophages obtained from the WT mice (Figure 5C). Consistent with these findings, immunofluorescence analyses confirmed that Cxcl1 and G-CSF were highly expressed in the heart after IR and that >50% of F4/80⁺ macrophages expressed Cxcl1 and G-CSF (Figure 5D and 5E and Figure VIII in the online-only Data Supplement). In addition, the expression of both chemoattractants was significantly decreased in D1KO mice compared with the WT mice. The majority of the Ly-6G⁺ neutrophils recruited to the heart after IR injury constitutively expressed Cxcr2 and G-CSFR, the main Cxcl1 and G-CSF receptors, which mediate neutrophil recruitment. Furthermore, we determined that neutrophil influx into the heart was considerably reduced in D1KO mice compared with the WT mice after IR, which was accompanied by a decrease in the expression of both Cxcr2 and G-CSFR (Figure 5B, 5F, and 5G and Figure IX in the online-only Data Supplement).

To explore the signaling pathway most likely activated by Dectin-1 that is involved in IR injury, we compared the expression of phosphorylated (p) spleen tyrosine kinase (Syk), p-p65, p-p38, p-Erk, and p-Jnk, and their total protein levels in both WT and D1KO mice. As shown, these pathways are activated during IR; however, only Syk, nuclear factor-κB (NF-κB), and p38-associated pathway expression were significantly reduced in D1KO mice (Figure 5H and 5I). Furthermore, we used specific inhibitors to investigate whether Dectin-1, Syk, NF-κB, and p38 are involved in the Cxcl1 and G-CSF production stimulated by the Dectin-1 agonist Curdlan AL in bone marrow–derived macrophages. Initially, bone marrow–derived macrophages derived from WT and D1KO mice were stimulated with Curdlan AL, and the obtained results demonstrated that the expression and secretion of Cxcl1 and G-CSF were significantly enhanced in response to Curdlan AL; however, their expression was greatly reduced in D1KO macrophages (Figure XA and XB in the online-only Data Supplement). Similar effects were confirmed in WT bone marrow–derived macrophages treated with Curdlan AL in the presence or absence of a Dectin-1 antibody to block Dectin-1 signaling (Figure XC and XD). Both R406, a specific inhibitor of Syk, and PDTC, a specific inhibitor of NF-κB, were shown to inhibit the expression and secretion of Cxcl1 and G-CSF, indicating that these molecules are involved in the Dectin-1–mediated chemoattractant expression pathway (Figure XE–XH in the online-only Data Supplement). However, the p38-associated mitogen-activated protein kinase pathway was shown not to be involved in this process; the p38 inhibitor SB203580 did not affect the expression or secretion of Cxcl1 and G-CSF (Figure XI and XJ in the online-only Data Supplement). Therefore, Dectin-1 appeared to contribute to Cxcl1 and G-CSF expression in macrophages through the Dectin-1/Syk/NF-κB signaling pathway.

Dectin-1–Induced IL-23/IL-1β Production Was Shown to Be Essential for the Expression of IL-17A by γδT Cells

Because Dectin-1 was reported to induce Th17 differentiation during fungal infection¹² and one of the main functions of IL-17A is neutrophil recruitment,¹³ we therefore examined Dectin-1–induced IL-17A expression and the regulation of neutrophil influx. Toward this end, we first characterized the IL-17A cell source in the mouse hearts after IR. Consistent with our previous findings,¹³ IL-17A was shown to be produced mainly by γδT cells, but not CD4⁺ (Th17) or CD8⁺ T cells (Figure 6A and 6B). IR injury resulted in a considerable increase in IL-17A production in the mouse hearts, which was shown to be significantly suppressed in D1KO mice (Figure 6C).

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We previously demonstrated that both IL-23 and IL-1β are required for IL-17A production from γδT cells¹³; thus, here we investigated whether IL-23/IL-1β mediated Dectin-1 induction of IL-17A production. mRNA levels of IL-23p19, IL-23p40, and IL-1β were significantly decreased in the sorted macrophages obtained from the IR hearts of D1KO mice compared with those in the hearts of WT mice (Figure 6D). Furthermore, anti–IL-23 receptor and anti–IL-1 receptor type I antibodies led to a decrease in IL-17A production from T cells and to neutrophil influx into the heart after IR (Figure 6E and 6F). Moreover, the neutralization of IL-17A in vivo was shown to lead to a significant reduction in the expression of Il-17A target genes Cxcl1 and Csf3 (G-CSF)¹⁴ in the heart after IR (Figure 6G).

Dectin-1 Activation Exacerbated Whereas Dectin-1 Inhibition Protected Against Myocardial IR Injury

To further extend functional analyses and to translate our findings to a clinical setting, we used 2 complementary approaches. We treated the myocardial IR-induced mice systemically with the Dectin-1 agonist Curdlan AL or Dectin-1–neutralizing antibody (anti–Dectin-1) before ischemia. The ratio of AAR to LV area was shown to be the same in all experimental groups, indicating that the ligature was reproducibly performed at the same level of the left anterior coronary artery. We found that the ratio of the infarct area to the AAR was significantly higher in mice treated with Curdlan AL than in those treated with PBS (53.43±2.73% versus 42.74±2.87%, respectively) and significantly lower in mice treated with Dectin-1 antibody than in those treated with the isotype alone (27.06±1.51% versus 42.34±1.99%, respectively) at day 2 after IR (Figure 7A and 7B). However, these effects were diminished in D1KO mice after IR injury, when the animals were treated with Curdlan AL or anti–Dectin-1 antibody (Figure XI in the online-only Data Supplement).

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Furthermore, Curdlan AL treatment resulted in a significant decrease in EF (31.82±0.84% in Curdlan AL–treated mice, 40.35±0.99% in PBS-treated mice), along with the increase in LVEDV and LVESV 4 days after the reperfusion, indicating the deterioration of cardiac function. Consistent with the infarct size, a notable improvement in cardiac function, as shown by the increase in EF (50.62±0.82% versus 39.31±1.17%, respectively) and the decrease in LV volumes (LVEDV and LVESV), was observed in the anti–Dectin-1 antibody–treated group compared with the isotype-treated group (Figure 7C and 7D and Table V in the online-only Data Supplement). These results were confirmed by hemodynamic measurements. LVEDP values were shown to be higher (13.5±1.2 mm Hg versus 8.8±0.6 mm Hg, respectively) whereas LV dP/dtmax (2631±310 mm Hg/s versus 3764±253 mm Hg/s, respectively) and dP/dtmin (−2935±293 mm Hg/s versus −3578±187 mm Hg/s, respectively) were lower in the Curdlan AL−treated group compared with the control group. Conversely, Dectin-1 inhibition led to an increase in dP/dtmax (4829±212 mm Hg/s versus 3842±268 mm Hg/s, respectively) and dP/dtmin (−4413±255 mm Hg/s versus −3698±214 mm Hg/s, respectively) and a decrease in LVEDP (4.4±0.9 mm Hg versus 8.5±0.8 mm Hg, respectively) compared with the control group (Table V in the online-only Data Supplement). However, these parameters derived from echocardiographic analyses did not significantly change after myocardial IR injury in D1KO mice treated with the Dectin-1 agonist and blocking antibody (Table V in the online-only Data Supplement).

Dectin-1⁺ Monocytes Were Increased in Patients With MI

Last, we addressed the relevance of our experimental findings to the human disease. Initially, we analyzed human heart samples after acute MI. We observed an abundance of Dectin-1⁺ cells in the infarct area of the MI hearts compared with the control, healthy tissue, which was accompanied by a profuse CD68⁺ macrophage and myeloperoxidase-positive neutrophil infiltration (Figure XIIA and XIIB in the online-only Data Supplement). Further analyses revealed that Dectin-1 was expressed mainly on CD68⁺ macrophages (Figure XIIC in the online-only Data Supplement). The expression of Dectin-1 was also significantly increased in patients with MI who had a large scar compared with those with a smaller scar, correlated with the severity of cardiac fibrosis (Figure XIID and XIIE in the online-only Data Supplement).

Next, we evaluated the levels of blood Dectin-1⁺ cells using flow cytometry in patients with ST-segment–elevation MI (STEMI) (n=90), including 45 patients with preserved LVEF and 45 patients with impaired LVEF. In addition, stable patients with normal coronary artery were enrolled as control subjects matched for age and sex (n=45). Patients with STEMI exhibited diabetes mellitus and dyslipidemia more often than control subjects. Compared with the control subjects, patients with STEMI had higher white blood cell, high-sensitivity C-reactive protein, and NT-proBNP (N-terminal pro-b-type natriuretic peptide) levels. In addition, patients with STEMI and reduced LVEF showed significantly poorer general condition compared with the group with MI and impaired LVEF (Table VI in the online-only Data Supplement). Monocytes can be classified as proinflammatory CD14⁺⁺CD16⁻ monocytes, intermediate CD14⁺⁺CD16⁺ monocytes, and nonclassic CD14⁺CD16⁺ monocytes (Figure XIII in the online-only Data Supplement).¹⁵ Flow cytometric analyses showed that the numbers of CD14⁺⁺CD16⁻ monocytes, CD14⁺⁺CD16⁺ monocytes, and neutrophils were significantly higher in patients with STEMI than in the control subjects and were also higher in those with impaired LVEF (Figure 8A, 8B, and 8D), whereas the number of natural killer cells was reduced in patients with STEMI and reduced LVEF, and CD14⁺CD16⁺ monocytes were comparable among the groups (Figure 8C and 8E). Furthermore, we observed that Dectin-1 was highly expressed in CD14⁺⁺CD16⁻ and CD14⁺⁺CD16⁺ monocytes, and relatively lower in CD14⁺CD16⁺ monocytes, but not in CD66b⁺ neutrophils and CD56⁺ natural killer cells (Figure XIII in the online-only Data Supplement). The percentages and numbers of Dectin-1⁺CD14⁺⁺CD16⁻ and Dectin-1⁺CD14⁺⁺CD16⁺ monocytes were significantly higher in patients with STEMI compared with the control subjects and were markedly elevated in those with reduced LVEF compared with patients with preserved cardiac systolic function. However, the numbers of Dectin-1⁺CD14⁺CD16⁺ monocytes, Dectin-1⁺ neutrophils, and Dectin-1⁺ natural killer cells did not differ among these groups (Figure 8A–8E).

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In addition, quantitative real-time polymerase chain reaction was performed in the whole peripheral blood of these subjects. As Figure 8F shows, the expression level of Dectin-1 was significantly elevated in patients with MI, especially those with impaired LVEF, compared with the control group, which was similar to the pattern of IL-6 expression. Simple correlation analysis further indicated that the expression of Dectin-1 was markedly associated with that of IL-6 (r=0.889, P<0.001; Figure 8G).

Discussion

In this study, we demonstrated a crucial role of Dectin-1 in the mediation of the myocardial IR injury in mice and humans. Myocardium-infiltrating macrophages, but not neutrophils, were shown to express Dectin-1, whereas Dectin-1 knockout or inhibition with anti–Dectin-1 antibody considerably ameliorated IR injury, leading to the reduction of cardiomyocyte apoptosis, associated with a reduced M1 macrophage polarization and Ly-6C⁺ monocyte and neutrophil infiltration. The activation of Dectin-1 with its agonist induced the opposite effects in mice. Furthermore, Dectin-1 contributed to neutrophil recruitment through the regulation of Cxcl1 and G-CSF expression, whereas Dectin-1–dependent IL-23/IL-1β production was demonstrated to be essential for IL-17A expression by γδT cells, which affected neutrophil recruitment and myocardial IR injury (Figure XIV in the online-only Data Supplement). Thus, Dectin-1 induced macrophage polarization and facilitated neutrophil infiltration by mediating chemokine expression and γδT cell activation, leading to myocardial IR injury.

Research by our group and others suggests that components of both innate and adaptive immunity contribute to IR injury.^4,16 IR triggers a vigorous immune response, augmented by the generation and release of various inflammatory cytokines and chemokines, which ultimately exacerbate myocardial injury.^2,4,17 Activation of the immune response occurs mainly when danger signals released from the ischemic and necrotic cardiomyocytes (damage-associated molecular patterns) interact with PRRs.¹⁸ The Toll-like receptor 2, Toll-like receptor 4, and NOD-like receptor P3 inflammasome have been reported to be critically involved in cardiac IR injury.^18–20 In an attempt to explore other PRRs associated with cardiac ischemia injury, we previously isolated cardiac macrophages at different time points after MI and performed microarray analyses.⁹ The obtained results demonstrated that Dectin-1 is highly expressed by macrophages in the early phase after MI, suggesting that it may play a role in ischemic diseases. Myocardial IR was suggested to lead to an increase in the number of Dectin-1⁺ macrophages, which explained the elevated myocardial Dectin-1 expression in the heart, suggesting that Dectin-1 may be critically involved in macrophage-associated inflammation and IR injury. In addition, Dectin-1 was recently shown to be involved in the regulation of autoimmunity, allergies, and homeostasis.⁶ For example, treatment with the Dectin-1 ligand β-glucan is able to induce local inflammation and arthritis in susceptible mice.²¹ However, to date, very little is known about the roles of Dectin-1 in myocardial IR injury; therefore, we explored these roles using a mouse model. As shown previously, we observed a significantly elevated Dectin-1 level in the myocardium during this process, beginning as early as 12 hours after the initiation of IR, peaking at 24 hours, and decreasing thereafter, suggesting that Dectin-1 is involved in the early stages of myocardial IR injury. Using gain- and loss-of-function strategies, we demonstrated that the activation and inhibition of Dectin-1 had opposite effects on the myocardial IR injury.

Ischemia-triggered cell necrosis in myocardium results in the release of necrotic products and cellular constituents such as heat shock proteins and nuclear DNA-binding protein high-mobility group box, which are capable of activating PRR signaling involved in the ischemic injury.¹⁸ Mincle, belonging to the C-type lectin receptor family, was shown to induce proinflammatory responses and to contribute to the development of ischemic disease after sensing SAP130, released by dead cells.²² However, Dectin-1 endogenous ligands or damage-associated molecular patterns involved in ischemia remain unknown, although some investigators have proposed that vimentin may serve as an endogenous activating ligand for Dectin-1 in atherosclerosis.²³ Therefore, further investigations are required to determine the nature of the ligands involved and how they interact with Dectin-1 during myocardial IR injury.

Dectin-1 was reported to be highly expressed on the populations of myeloid cells (monocyte/macrophage and neutrophil lineages); however, lower levels of expression of this molecule can be detected on dendritic cells and a subpopulation of T cells.²⁴ In addition, inflammatory macrophages exhibit the highest surface expression levels of Dectin-1, indicating the role of this receptor in the inflammatory process. In contrast, resident macrophages expressed much lower levels of Dectin-1 on the cell surface.²⁴ In line with these findings, we found that Dectin-1 is expressed exclusively by CD11b⁺ myeloid cells, whereas lymphocytes did not express this molecule. Further analyses revealed that proinflammatory macrophages, but not neutrophils, represent the main population expressing Dectin-1. Furthermore, by using a BMT strategy, we confirmed that Dectin-1 expressed on bone marrow–derived cells mediates myocardial IR injury, which implies that targeting Dectin-1 on macrophages may constitute a beneficial therapeutic approach for the prevention of IR injury.

During myocardial IR injury, macrophages were shown to be the predominant cell type and to display functional heterogeneity, with proinflammatory macrophages (M1 macrophages) infiltrating initially, followed by a second wave of anti-inflammatory macrophages (M2 macrophages), which is analogous to the previously described recruitment of proinflammatory and anti-inflammatory monocytes.^4,25 M1 macrophages express TNF-α, iNOS, IL-1β, and IL-6, and they induce a strong proinflammatory reaction and contribute to myocardial IR injury. In contrast, M2 macrophages express IL-10 and arginase-1 and -2 instead of iNOS, depleting arginine stores and producing polyamine and proline (instead of nitric oxide), which are important for cell differentiation and collagen production, respectively.^2,4,13,25 Recently, Liu et al²⁶ reported that the activation of Dectin-1 with the yeast-derived particulate β-glucan converts polarized M2 bone marrow–derived macrophages and immunosuppressive macrophages to M1-like phenotype, expressing proinflammatory cytokines such as TNF-α, iNOS, IL-1β, and IL-6, which lead to a reduction in tumor progression. This effect is associated with the metabolic reprograming of macrophages, and this process is mediated through the Dectin-1–dependent canonical Syk/Card9/Erk pathway. A different study showed that Dectin-1–deficient macrophages exhibit an M2 phenotype in response to Paracoccidioides brasiliensis infection, resulting in an impaired fungicidal ability, low nitric oxide production, and elevated synthesis of IL-10.²⁷ Here, we showed that ischemia activated Dectin-1 in the heart, leading to M1 macrophage polarization and promoting relatively higher expression of proinflammatory cytokines and lower CD206 expression, which is at least partially involved in the expansion of IR injury. However, although we demonstrated that during IR, Dectin-1 affects Syk-, NF-κB–, and p38-associated pathways, which regulate neutrophil recruitment through the regulation of the expression and secretion of Cxcl1 and G-CSF after IR, further studies are necessary to examine the signaling pathways involved in Dectin-1–induced M1 macrophage activation during myocardial ischemia. Moreover, the use of a simple M1/M2 model to describe macrophage polarization in vivo is debatable, and further studies should elucidate the exact macrophage phenotype and gene expression profile altered by the Dectin-1 signaling pathway in myocardium in response to ischemia.

Neutrophil recruitment and infiltration into the infarcted area play a critical role in myocardial damage after IR.^2,16 Through the generation of reactive oxygen species and proteolytic enzymes, neutrophils injure surrounding cardiomyocytes.¹⁶ Neutrophil production, chemotaxis, and trafficking may be strongly regulated by ELR⁺CXC chemokines and G-CSF during inflammatory diseases,^28,29 and the importance of ELR⁺CXC chemokines in myocardial IR has been demonstrated in vivo.³⁰ Notably, Dectin-1 was shown to regulate the expression of Cxcl1/KC and G-CSF in a Syk- and NF-κB–dependent manner, leading to neutrophil recruitment in response to pathogen infection.^31,32 Here, we found that the absence of Dectin-1 considerably decreases macrophage Cxcl1 and G-CSF expression and neutrophil infiltration in mouse IR myocardium. The infiltrated neutrophils showed high expression of Cxcr2 and G-CSFR in heart, indicating that Dectin-1–dependent Cxcl1/G-CSF expression is required for neutrophil infiltration and myocardial IR injury. Both in vivo and in vitro studies further revealed that Syk- and NF-κB–associated pathways may be involved in these processes.

In our previous study, we demonstrated that IL-23 and IL-1β induce IL-17A production from cardiac γδT cells, which promotes the infiltration of neutrophils and M1 macrophage activation, aggravating the rate of cardiomyocyte death and enhancing fibroblast proliferation and profibrotic gene expression during MI.¹³ In accordance with our findings, another group, on the basis of our research, demonstrated that IL-17A–mediated cardiomyocyte apoptosis, through the regulation of the Bax/Bcl-2 ratio, induces CXC chemokine–mediated neutrophil migration and promotes neutrophil–endothelial cell adherence via the induction of endothelial cell E-selectin and intercellular adhesion molecule-1 expression after myocardial IR.³ Previous studies demonstrated that the expression of IL-23 and IL-1β is dependent on Dectin-1 expression in vivo,^26,27 and Dectin-1 was reported to regulate the production of Th17 cytokines and to induce the migration of neutrophils to the infected tissues.²⁷ Furthermore, IL-17A modulates neutrophil behavior through cytokines that promote polymorphonuclear cell expansion and survival (G-CSF), as well as neutrophil chemoattractants (Cxcl1, Cxcl2, and Cxcl5).¹⁴ In the present study, we showed that IL-17A is produced mainly by γδT cells, which are dependent on Dectin-1–induced IL-23 and IL-1β expression in cardiac macrophages. Moreover, the inhibition of IL-23 and IL-1β attenuated neutrophil infiltration in the heart after IR. Taken together, these results, along with our previous findings, indicate that Dectin-1 contributes to myocardial IR injury at least partially through regulation of the IL-23/IL-1β/IL-17A immune axis and neutrophil infiltration to the myocardium. Thus, Dectin-1 could induce macrophage polarization toward the M1 phenotype, directly aggravating myocardial injury by releasing proinflammatory cytokines, including TNF-α, IL-1β, and IL-23, as well as indirectly by mediating neutrophil infiltration. The latter process depended both on the γδT cell activation through Dectin-1–dependent IL-23/IL-1β expression and on the secretion of the chemokines CXCL1 and G-CSF.

Translation of animal findings into human pathology is often questioned. To decipher whether Dectin-1 has similar roles in humans, we examined Dectin-1 expression in the heart and peripheral blood samples obtained from patients with MI and control subjects. We observed that Dectin-1⁺ cells abundantly infiltrated in the myocardium from patients with MI, with Dectin-1 expressed mainly on macrophages. Furthermore, in line with the findings obtained with animal models, Dectin-1 was shown to be highly expressed by human circulating proinflammatory CD14⁺⁺CD16⁻ and CD14⁺⁺CD16⁺ monocytes and positively correlated with the proinflammatory cytokine IL-6. However, its expression was much lower on neutrophils and natural killer cells. Further analyses revealed that its expression level significantly increased in patients with MI and reduced LVEF compared with those with preserved LVEF, implying that Dectin-1 may constitute an important mediator during MI in humans. These data are consistent with results of previous studies in which an elevated circulating IL-6 level in the setting of early acute MI was shown, and the simultaneous elevation of IL-6 and IL-10 levels distinguishes patients with STEMI with worse clinical outcomes from other patients with STEMI.^33–35

Conclusions

Here, we demonstrated that Dectin-1 expressed by macrophages plays a pathogenic role during myocardial IR injury by inducing macrophage polarization toward the M1 phenotype and neutrophil infiltration. These data suggest a novel Dectin-1–dependent pathway in the heart through which the immune system may influence myocardial IR injury. Control of Dectin-1 activation may therefore be beneficial for the minimization of IR-induced myocardial damage.

Footnotes