Introduction
Myocardial infarction (MI) and the resulting complications are a major cause of death worldwide. Following MI, circulating blood monocytes respond to chemotactic factors, migrate into the infarct myocardium, and differentiate into macrophages. At the injury site, macrophages remove necrotic cardiac myocytes and apoptotic neutrophils; secrete cytokines, chemokines, and growth factors; and modulate phases of the angiogenic response. As such, macrophage is a primary responder cell type that is involved in the regulation of post-MI wound healing at multiple levels. Two phases of infiltration are defined after MI. In the first few days after injury, inflammatory monocytes and classical M1 macrophages rapidly invade the wound to defend against pathogens, phagocytose, and lyse debris, and thus pave the way for tissue regeneration [
1]. During subsequent healing, classical macrophages retreat and give way to M2 macrophages, which exhibit a less inflammatory panel of functions that supports tissue repair and regeneration [
2-
4]. Recent studies have indicated that anti-inflammatory strategies targeting inflammatory monocyte/macrophage subsets could reduce excessive inflammation and improve cardiovascular outcomes [
5-
8]. Thus, identification of targets that modulate macrophage phenotypes and functions may lead to development of novel therapeutic approaches for MI.
In an effort of searching for molecules involved in macrophage polarization, we established a phenotypic screening assay and screened molecules that are able to switch M1 to M2 using pooled shRNA library. Collapsin response mediator protein-2 (CRMP2), a multifunctional adaptor protein first described in the CNS and thoroughly studied in neurons [
9-
11], was identified as a novel protein potentially involved in macrophage polarization. CRMP2 belongs to a family of five homologous members and was first identified as a mediator of semaphorin-induced growth-cone collapse [
10]. Downstream of semaphorin signal, they reorganize the cytoskeleton by controlling microtubule assembly [
12-
14], thereby playing a crucial role in axonal outgrowth and neurite extension [
10]. In recent years, several observations have shown that CRMP2 is present in the immune system and plays a critical role in T lymphocyte polarization and migration [
15-
17]. CRMP2 has the ability to bind to the cytoskeletal elements tubulin and vimentin redistributed at the uropod, the flexible structure of T cells, and RNA mediated crmp2/dpysl2 gene silencing and blocking antibody strongly reduces T cell polarization (uropod formation) and migration. Conversely, increased CRMP2 expression promotes uropod formation and the migration of transfected lymphocytes [
16]. Thus, CRMP2 is a key player in cell behavior within a wider field than just the nervous system.
In the present study, we observed that CRMP2 was expressed in a significantly higher level in M1 macrophages than M2 macrophage subsets, and knockdown of CRMP2 with RNA interference (RNAi) in M1 macrophages resulted in a significant decrease in M1 gene expression and increase in M2 gene expression. High level of CRMP2 was also observed in the macrophages infiltrated in the infarct area 3 days post MI in both wildtype (WT) and ApoE−/− mice, and the expression of CRMP2 retained in the infiltrated macrophages of ApoE−/− mice but not in that of WT mice 10 days after MI. In ApoE−/− mice with MI, nanoparticle-mediated delivery of CRMP2 siRNA to wound macrophages efficiently suppressed expression of CRMP2 in vivo, associated with reduced expression of inflammatory M1 macrophage markers, increased resolution of inflammation, accelerated infarct healing, and attenuated fibrosis, development of post-MI heart failure and mortality.
Materials and methods
Animal models
C57BL/6 J and B6.129P2-Apoetm1Unc/J (ApoE
−/−) mice used in this study were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). ApoE
−/− mice were fed on a high-cholesterol diet for 6 months (Harlan Teklad, 0.2% total cholesterol). Myocardial infarction was induced by permanent coronary ligation [
18]. Briefly, animals were anesthetized using a mixture of ketamine, xylazine and atropine (100, 20 and 1.2 mg/kg, respectively, i.p.) and mechanically ventilated. Under a surgical microscope, left thoracotomy was performed to expose the heart. The left coronary artery was identified and ligated with a 7–0 silk suture at a level approximately 2 mm below the edge of the left auricle. Sham operation was also performed without ligating the coronary artery. After surgery mice were monitored daily for 4 weeks and autopsy was performed on all mice found dead to identify the cause of death such as post-MI cardiac rupture or heart failure, as described previously [
18]. Some mice were sacrificed at 6 and 24 h, 3, 7 and 14 days, and 4 weeks, respectively, following MI. Infarct and non-infarct myocardium were separated and snap frozen in liquid nitrogen and stored at −80°C for molecular assays. Further, some hearts were fixed in 10% formalin or fresh frozen for embedding with OCT for histological analyses. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Ningxia Medical University.
Culture of bone marrow-derived macrophages
Bone marrows were obtained from 8–12 week old C57BL/6 mice, and bone marrow-derived macrophages (BMDMs) were cultured using a previously described protocol [
19]. In brief, bone marrow cells were flushed out from mouse tibias and femurs with Dulbecco’s modification of Eagle’s medium (DMEM; Gibco BRL, Shanghai, China) containing 100 μg/ml primocin (Invivogen, San Diego, CA, USA) and then filtered through 100-μm cell strainer (BD, Shanghai, China). Red blood cells were removed with lysis buffer (0.75% NH4Cl, 0.02% Tris–HCl, pH 7.2) on ice for 15 min. Bone marrow cells were cultured in a DMEM supplement with 15% fetal calf serum (Sigma-Aldrich, Shanghai, China) and 15% L929 conditioned medium and seeded in 75-mm2 flasks at 37°C in 5% CO2 incubator for 5 days. L929 is a murine fibroblast cell line that produces M-CSF and has been used widely for macrophage differentiation studies [
20].
Activation of BMDMs
BMDMs were seeded in six-well plates at a density of 1 × 106 cells/well for overnight. Cells were then activated as follows: M1, IFN-γ (100 ng/ml; R&D Systems, Shanghai, China) and LPS (50 ng/ml, Sigma-Aldrich); M2a, IL-4 (20 ng/ml; R&D Systems, Shanghai, China); M2b, immune complex (150 μg/ml anti-chicken egg albumin (ovalbumin) monoclonal antibody preincubated with 15 μg/ml ovalbumin at 37°C for 30 min, Sigma-Aldrich) and LPS (50 ng/ml); and M2c, IL-10 (20 ng/ml, R&D Systems). Seven and 24 h later, cells were harvested for total RNA or protein extraction. Trypan blue staining did not reveal significant cell death in activated BMDMs at both time points. To collect the supernatants, cells were activated with appropriate stimuli for 7 h. The cells were then washed with phosphate-buffered saline (PBS) and incubated with serum-free DMEM. Forty-eight hours later, supernatants were collected and stored in −80°C until use.
Construct and RNA interference
Three CRMP-2 siRNAs (siCRMP-2-1, siCRMP-2-2 and siCRMP-2-3) were designed to target mouse CRMP-2 mRNA (NM_009955) sequences: 5’-ACUCCUUCCUCGUGUACA-3′, 5’-GAUGGGUUGAUCAAGCAA-3′ and 5’-ACTCCTTCCTCGTGTACAT-3’ respectively. A non-targeting siRNA was used as a negative control for all siRNA transfection experiments. All siRNAs were synthesized by Shanghai GenePharma (Shanghai, China). The efficacy and specificity of siRNAs were determined as described previously [
21].
Nanoparticles were prepared with the cationic lipid C12-200, disteroylphosphatidyl choline, cholesterol, and PEG–DMG using a spontaneous vesicle formation formulation procedure [
22]. In brief, lipids were dissolved in 90% ethanol solution and mixed with siRNA solution (25 mM citrate, pH 3 ratio) at fixed speed (1:1 ratio) and diluted immediately with PBS to final 25% ethanol. The ethanol was then removed and the external buffer replaced with PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4, pH 7.5) by dialysis. The final lipid:siRNA weight ratio was ∼ 7:1. Particle size and zeta potential were determined using a Malvern Zetasizer NanoZS (Malvern, UK). siRNA content was determined by ion exchange HPLC (Agilent) assay using DNAPac Pa200 column (Dionex Corporation Dionex, 260 nm, 55°C run at 2 mL/min). siRNA entrapment efficiency was determined by the Quant-iT RiboGreen RNA assay (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. For intravenous injection, mice were anesthetized and injected through the tail vein with 0.5 mg/kg of either siCRMP2 or control siRNA (siCON) incorporated into lipidoid nanoparticles (LNPs).
Plasmids and adenoviral infection
IRF expression construct was generated in the pENTR vector (Invitrogen) modified to contain the CMV promoter and IRES-linked GFP (pBent) as previously described [
23]. For delivery into mouse bone marrow-derived macrophages, IRF5/luciferase cassettes were excised and subcloned into the pBent vector, modified to contain CMV-driven GFP in the orientation opposite to the luciferase gene and recombined into pAD/PL DEST vector (Invitrogen) for adenovirus production. Adenoviral infections of mouse bone-marrow-derived macrophages were performed in 96-well plates in triplicate. The plates with serum-free RPMI medium 1640 containing the desired number of viral particles were centrifuged at 400 g for 30 min then placed at 37°C overnight. The next day the virus media were replaced with 100 μL of standard media and the cells were allowed to recover for 2 days before the application of experimental conditions.
Quantitative RT-PCR
Total messenger RNA (mRNA) was extracted using the RNeasy Micro Kit (Qiagen) according to manufacturer’s instructions. One microgram of mRNA was reverse transcribed using the high capacity RNA to cDNA kit (Applied Biosystems). TaqMan gene expression assays (Applied Biosystems) were used to quantify target genes. The relative changes were normalized to Gapdh mRNA using the 2-∆∆CT method.
Western blot analysis
Myeloid cells from heart tissue were isolated as described above, washed with ice-cold PBS and homogenized on ice using RIPA lysis buffer (Millipore) supplemented with complete protease inhibitor cocktail (Roche). Protein concentration was measured using BCA assay (Pierce). Samples of 15 μg were loaded on 10% SDS-PAGE and transferred onto PVDF membranes (Bio-Rad). Membranes were blocked with 5% non fat dry milk in TBS-Tween 0.1% and incubated with primary antibodies against the specific protein and peroxidase-coupled secondary antibodies. β-actin was used as control. Signals were visualized with enhanced chemiluminescence detection system (ECL Plus, Amersham Life Science), and densitometric analysis was performed with ImageJ 1.40 g (National Institutes of Health). The following antibodies were used: rabbit polyclonal anti-CRMP2 (Abcam), rabbit polyclonal anti-IRF5 (Abcam), mouse monoclonal anti-IRF4 (eBioscience), rabbit polyclonal anti-IRF3 (Abcam), rabbit monoclonal anti-CD86 (Abcam), rabbit polyclonal anti-CD163 (Santa Cruz Biotechnology).
Histology
To eliminate blood contamination, hearts were perfused with ice-cold PBS after mice were euthanized. Hearts were removed, rinsed in PBS, embedded in O.C.T. compound (Sakura Finetek), and frozen in an isopentane bath on dry ice. For immunofluorescence staining, sections (5 μm) were stained with mixture of antibodies, including mouse monoclonal anti-CD11b antibody (clone M1/70, BD Biosciences) and a rabbit polycolonal anti-iNOS (Abcam), mouse monoclonal anti-CD11b antibody (BD Biosciences) and a rabbit polycolonal anti-IGF-1 (Abcam), or mouse monoclonal anti-CD11b antibody (BD Biosciences) and a rabbit polycolonal anti-CRMP2 (Abcam), followed by incubation with a mixture of Alexa Fluor® 488 Goat anti-mouse IgG and Cy3-conjugated anti-rabbit IgG. The slides were cover slipped using a mounting medium with DAPI (Vector Laboratories, Inc.) to identify nuclei. Images were observed and captured using Nikon Eclipse 80i with a Cascade Model 512 B camera (Roper Scientific). For immunohistochemistry, histology of the heart was performed on day 7 after MI in ApoE−/− mice. Frozen sections (5 μm) were stained for antibodies against CD11b (BD Biosciences), CD86 (Abcam), and CD206 (Abcam). The appropriate biotinylated secondary antibodies, ABC kit (Vector Laboratories, Inc.) and AEC substrate (Dako) were used for color development, and all the sections were counterstained with Harris hematoxylin. The slides were scanned by a digital slide scanner, NanoZoomer 2.0-RS in 40x high resolution mode (Hamamatsu, Japan). The positive area was quantified using IPLab (version 3.9.3; Scanalytics, Inc.) and analyzing five high power fields per section and per animal.
Postmortem histological determination of scar area
Mice were euthanized by cervical dislocation after anesthesia with 5% isoflurane for histological assay at 4 weeks after MI [
24]. Hearts were embedded in paraffin after being fixed in 4% paraformaldehyde. Serial sections (5 mm thickness) were performed Masson’s trichrome stain to detect scar area and fibrosis in cardiac muscle. Computerized morphometry was used to calculate the scar extent as the ratio of scar and total left ventricular area using Imaging Pro Plus software.
Echocardiographic studies of cardiac function
Echocardiography was performed to assess the cardiac function after MI in a blinded manner [
25]. At 2 days post operation (POD) and weekly until sacrificed, Mice were anesthetized (2% isoflurane and oxygen) and put in a supine position. Both two-dimensional and M-mode images were recorded using a 30-MHz transducer. Left ventricular systolic dimension (LVDs), left ventricular diastolic dimension (LVDd), anterior wall thickness (AWT) and posterior wall thickness (PWT) were measured to calculate left ventricular ejection fraction (LVEF) and fractional shortening (FS) as an average of three beats.
Statistics
Data are expressed as mean ± sem. Analyses were performed using Prism 6.0a (GraphPad Software Inc.). The group means were compared using a Student’s t-test (for 2 groups) and ANOVA, followed by Bonferroni post-tests (for > 2 groups). P values of <0.05 indicate statistical significance.
Discussion
With the recent insight into the molecular mechanisms governing macrophage heterogeneity, polarization and function [
31], it has become feasible to modulate macrophage actions in interventions that might optimize healing of injured tissues. Identification of appropriate targets that modulate macrophages phenotypes is critical for this goal. In the present study, we provided the first evidence that CRMP2 is expressed in macrophages and its expression depends on the activated status of the cells. CRMP2 is predominantly expressed in M1 macrophages, and plays a role in macrophage polarization to M1 as silencing CRMP2 results in switch of macrophages from M1 to M2 not only
in vitro but also in atherosclerotic mice with MI. Thus, modulation of CRMP2 expression may promote inflammation resolution and improve infarct healing.
Macrophages are key mediators of the immune response during inflammation. Plasticity and functional polarization are hallmarks of macrophages that result in the phenotypic diversity of macrophage populations [
32]. Though many transcription factors, including PU.1, C/EBPβ, Runx1 and IRF8, are involved in lineage-specific transcriptional regulation during macrophage differentiation [
33], only a small proportion of the macrophage transcriptome is altered by cell polarization [
34]. Among the transcription factors, IRF5 is defined to determine commitment to the M1 macrophage lineage [
23]. Another member of the IRF family, IRF4, known to inhibit IRF5 activation by competing for interaction with the adaptor Myd88, is reported to control the expression of prototypical mouse M2 macrophage markers [
28]. We found that knockdown of CRMP2 reduced the expression of a number of M1 genes, including ccr7, cox2, tnf-α, cd86, il12b and cxcl10, and increased the expression of several M2 genes, including ym-1, arg-1, and il-10. These were associated with down-regulation of IRF5, without affecting the expression of IRF3 and IRF4. We further demonstrated that CRMP2-knockdown-induced M1 to M2 switch was reversed by overexpression of IRF5 in M1 macrophages. Therefore, it is postulated that CRMP2 may play a role in modulating macrophage polarization to M1 through, at least in part, regulating IRF5 expression. However, the underlying mechanism remains to be elucidated. It is postulated that CRMP2, which is localized in both cytoplasm and nucleus, may be directly involved in the transcriptional regulation of IRF5 or physically interact with IRF5 thereby stabilizing IRF5. These hypotheses need to be confirmed in future studies.
Enforcing the natural transition of M1 toward M2 macrophages in wounds may thus usher in resolution of inflammation and speed healing, especially if acute wound inflammation exists in the setting of an underlying chronic inflammatory disease. A prolongation of the inflammatory phase of wound healing inhibits regenerative processes and may compromise tissue integrity. Coronary ligation in ApoE
−/− mice allows the study of MI in the context of pre-existing chronic inflammation [
29]. These atherosclerotic mice have impaired resolution of inflammation post-coronary ligation due to delayed M1 and M2 transition [
30], thereby having a higher risk of developing heart failure post-MI, possibly due to compromised infarct healing [
35]. We observed a significantly prolonged infiltration of M1 macrophages in the infarct of ApoE
−/− mice compared to that in wildtype mice. Intriguingly, the inflammatory macrophages in the infarct wound expressed CRMP2, which was localized at one pole outside of the nucleus.
To investigate the effect of CRMP2 knockdown on infarct healing in ApoE
−/− mice with MI, we used a lipidoid nanoparticle to deliver CRMP2 siRNA into the wound because employing the endocytic machinery of macrophages, intravenously administered nanoparticles are rapidly taken up by monocytes/macrophages, and accumulated in macrophages in atherosclerotic plaques [
36,
37], rendering inflammatory myeloid cells a prime target for
in vivo RNAi [
38-
40]. In ApoE
−/− mice with MI, nanoparticle-mediated delivery of siRNAs has been shown to efficiently knockdown the target genes in macrophages in the infarct wound [
18]. Consistently, we demonstrated that nanoparticle-mediated delivery of CRMP2 siRNA resulted in an efficient knockdown of CRMP2 in macrophages infiltrated in the wound. This was associated with a significantly decreased proportion of M1 but markedly increased proportion of M2 macrophages in the wound, whereas total macrophage percentage did not differ between the control and CRMP2 siRNA groups. We showed that in vivo knockdown of CRMP2 supported the resolution of inflammation, as numbers of inflammatory cells, including monocytes, neutrophils and macrophages were reduced. Moreover, CRMP2 RNAi decreased the extent of fibrosis, leading to an enhanced cardiac function recovery and decreased the mobility after MI.
In conclusion, we show that CRMP2 plays a role in macrophage polarization of M1 phenotype and CRMP2 RNAi resulted in a switch of M1 to M2 macrophages not only in vitro but also in ApoE−/− mice with MI, leading to an promoted inflammation resolution, enhanced cardiac function recovery and decreased mobility after MI. Although our study bears some clinical relevance, the detailed physiologic and pathologic functions of CRMP2 have not been extensively characterized. Further studies defining the exact underlying mechanisms are needed.
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Competing interests
There authors declare that they have no competing interests.
Authors’ contributions
L-SZ performed research design and studies for figures
1,
2 and
3; G-LZ formed studies for figures
4 and
5, QL performed studies for figure
6; S-CJ performed studies for figure
7; YW oversaw the whole project, provided scientific input, and completed the manuscript; D-MZ provided scientific and technical support for the animal studies and helped finish the manuscript. All authors read and approved the final manuscript.
Long-Shu Zhou and Guo-Long Zhao, Residents and Research associates, Department of Cardiovascular Surgery, General Hospital, Ningxia Medical University. Qiang Liu and Shu-Cai Jiang, Research Instructors, Department of Neurosurgery, General Hospital, Ningxia Medical University. Yun Wang, Professor and Director, Department of Cardiovascular Surgery, General Hospital, Ningxia Medical University. Dong-Mei Zhang, Professor and Director, Department of Anesthesiology, General Hospital, Ningxia Medical University.