Introduction
Cicatrization, or fibrosis, is a normal, inevitable physiological response, which occurs during natural post-burn healing, however, it is clinically challenging due to the severe effect on quality of life. Macrophages are important in wound healing and fibrosis (1). Lucas et al (2) found that eliminating macrophages delayed wound healing by decreasing granulation tissue growth and inhibiting re-epithelialization. By isolating and culturing macrophages from wounds, Sindrilaru et al (3) observed that different phenotypes were generated in response to various microenvironmental stimuli. Macrophages continuously express M1 (classic activation) and M2 (alternative activation) surface markers throughout wound healing, with the M1 and M2 types dominating at the beginning of the inflammatory response and at the repair phase, respectively (4,5). As profibrotic cells, M2 macrophages secrete cytokines, which activate the transformation of fibroblasts into myofibroblasts, thus promoting cicatrization by leading to the overgrowth of granulation tissues and deposition of extracellular matrix (ECM) (6).
Eukaryotic initiation factor 6 (eIF6), which regulates the initial translation of eukaryotes, binds to large ribosomal subunit 60S in cell nuclei to inhibit the formation of 80S ribosomes (7). At the beginning of protein translation, eIF6 is depolymerized from the 60S subunit through phosphorylation under the action of casein kinase 1-protein kinase C, which facilitates the binding between 40S and 60S subunits to form the 80S subunit, thereby initiating the translation process (8). Our previous study compared the wound healing outcomes of eIF6 wild-type (eIF6+/+) and knockout (IF6+/−) mice, and found that the latter had significantly more granulation tissue and collagen, indicating that eIF6 inhibited skin fibrosis (unpublished data). However, the mechanism by which eIF6 affects macrophages, wound healing and cicatrization remains to be elucidated. The present study aimed to identify the target gene by comparing the gene expression profiles of M2 macrophages in eIF6+/+ and eIF6+/− mice with microarrays. This may provide theoretical evidence for the role of eIF6 in inhibiting fibrosis, and a novel therapy strategy for mitigating scar repair and fibrosis in clinical practice.
Materials and methods
Experimental animals and reagents
A total of 24 male eIF6 wild-type (eIF6+/+) and knockout (eIF6+/−) C57BL/6 mice (8–10 weeks old; 15–20 g; Beijing HFK Bioscience Co., Ltd., Beijing, China) were used in the present study. The mice were maintained in a temperature (18–22°C) and humidity (50–60%) controlled environment with ad libitum access to food and water. eIF6+/+ and eIF6+/− mice were maintained in 12 h/12 h and 22 h/22 h light/dark cycles, respectively. Culture medium comprising RPMI 1640 and fetal bovine serum was purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Recombinant mouse interleukin-4 (IL-4) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The following monoclonal mouse antibodies were purchased from Abcam (Cambridge, MA, USA): Anti-F4/80-peridinin chlorophyll protein complex (PerCP; 1:1,200 dilution; cat. no. ab111101), anti-CD11b-fluorescein isothiocyanate (FITC; 1:900 dilution; cat. no. ab24874), anti-Cd11c-phycoerythrin (PE; 1:1,000 dilution; cat. no. ab155349), anti-CD206(MR)-FITC (1:1,000 dilution; cat. no. ab64693) and anti-CD16/32 (1:1,200 dilution; cat. no. ab25235). The RNeasy Mini kit (cat. no. 2183018A) was provided by Life Technologies; Thermo Fisher Scientific, Inc. The AMV3.0 Reverse Transcription kit was purchased from Takara, Bio Inc. (Otsu, Japan; cat. no. RR019A). Gene microarrays (Affymetrix GeneChip Mouse Gene1.0 ST Array) were purchased from Affymetrix (Santa Clara, CA, USA). The study was approved by the ethics committee of The First Hospital of Hebei Medical University (Shizjiazhuang, China).
Culture of peritoneal macrophages
The mice were sacrificed by cervical dislocation and the peritoneal cavity of each was opened following sterilization with 75% ethanol and repeatedly lavaged with 10 ml pre-cooled RPMI 1640 medium. The lavage fluid was collected into sterile centrifuge tubes placed on ice, and centrifuged at 4°C and 12,000 × g for 5 min to obtain the cell precipitate, which was resuspended with 2 ml RPMI 1640 medium, containing 10% fetal bovine serum. Subsequently, the cells were inoculated at a density of 5×106 onto 6-well plates and cultured in an incubator for 12 h at 37°C in an incubator containing 5% CO2, of which those exhibiting wall-adherence were identified as macrophages. The stimulated group was treated with 40 ng/ml IL-4 for 24 h to acquire M2 macrophages, and an equal volume of phosphate-buffered saline (PBS) was added to the non-stimulated group. All other conditions were identical.
Detection of macrophage purity
The cultured macrophages were digested with 0.5% trypsin (Sigma-Aldrich), counted at the density of 5×105/tube, washed once with PBS, resuspended in 100 µl PBS and incubated with 1 µl anti-CD16/32 for 10 min at 37°C to block the Fc receptor. The cells were then incubated with 1 µl anti-F4/80-Percp and 1 µl anti-CD11b-FITC at room temperature for 30 min, washed with 1 ml PBS to remove free antibodies, and resuspended in 0.3 ml PBS. Finally, the expression levels of F4/80 and CD11b on the cell surface were detected using an Attune Flow Cytometer (Applied Biosystems; Thermo Fisher Scientific, Inc.).
Detection of surface markers on M2 macrophages
The stimulated macrophages were digested with 0.5% trypsin, counted at a density of 5×105/tube, washed once with PBS, resuspended in 100 µl PBS and incubated with 1 µl anti-CD16/32 for 10 min to block the Fc receptor. Subsequently, the cells were incubated with anti-F4/80-Percp, anti-CD11c-PE and anti-CD206 (MR) -FITC (1 µl each) at room temperature for 30 min, washed with 1 ml of PBS to remove free antibodies, and resuspended in 0.3 ml PBS. Finally, the expression levels of F4/80, CD11c and CD206 on the cell surface were detected using flow cytometry.
Gene microarray analysis
The RNAs of the M2 macrophages from the eIF6+/+ and eIF6+/− mice were extracted and subjected to gene microarray analysis in triplicate. The detailed information is available at http://www.affymetrix.com.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA (1 g) was extracted from the macrophages using an RNeasy Mini kit and transcribed into cDNA. Then, qPCR was performed in a 10 µl reaction system using a 7700 Real-time Fluorescence Quantitative PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) to detect the expression levels of transforming growth factor-β1 (TGF-β1), arginase-1, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2) and vascular endothelial growth factor (VEGF). The total reaction volume for PCR (25 µl) contained 12.5 µl 2× Premix Ex Taq, 1 µl upstream and downstream primers, 1 µl cDNA and 9.5 µl ddH2O. The PCR thermal cycling conditions were as follows: 95°C for 10 min, 95°C for 10 sec, 60°C for 30 sec and 72°C for 20 sec, with 40 cycles in total. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) and the sequences were as follows: GAPDH, sense 3′-GGGGAAGGTGAAGGTCGGAGTC-5′ and antisense 3′-TCGCTCCTGGAAGATGGTGATG-5′; TGF-β1, sense 3′-TGGAAACCCACAACGAAATCTATGA-5′ and antisense 3′-TGGAAACCCACAACGAAATCTATGA-5′; arginase-1, sense 3′-CTCCAAGCCAAAGTCCTTAGAG-5′ and antisense 3′-GGAGCTGTCATTAGGGACATCA-5′; MMP-2, sense 3′-ACCTGAACACTTTCTATGGCTG-5′ and antisense 3′-CTTCCGCATGGTCTCGATG-5′; TIMP-2, sense 3′-GCAACCCCATCAAGAGGATTC-5′ and anti-sense 3′-GGGGCCGTGTAGATAAACTCG-5′; VEGF, sense 3′-GCCAGACAGGGTTGCCATAC-5′ and antisense 3′-GGAGTGGGATGGATGATGTCAG-5′. GAPDH was used as the internal reference, and the results were expressed using the 2−ΔΔCq method (9). All experiments were performed in triplicate.
ELISA
The supernatant obtained from the culture medium of macrophages following stimulation with IL-4 for 24 h was collected, and the expression levels of VEGF (cat. no. EK0506), MMP-2 (cat. no. EK0511) and TIMP-2 (cat. no. EK0322) were detected using ELISA kits (Boster Biological Technology Co., Ltd., Wuhan, China), in strict accordance with the manufacturer's protocol. All experiments were performed in triplicate.
Statistical analysis
All data were analyzed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) and expressed as the mean ± standard deviation. Inter-group comparisons were compared using a Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
Expression levels of surface molecules and determination of the purity of M2 macrophages
Following 24 h of IL-4 stimulation, the macrophages underwent marked morphological changes, including decreased dendritic branches, cell rounding and reduced refractive index (Fig. 1A and B). Flow cytometry showed that the expression of CD206 in the IL-4 stimulated group was significantly increased, compared with that in the non-stimulated group (Fig. 1C–E), whereas the change in the expression of CD11c was minimal (Fig. 1F and G). The purity of the M2 macrophages was 94.4% (Fig. 1H).
Function of M2 macrophages
Under in vitro culture conditions, the macrophages were induced into M2 macrophages in the present study through stimulation with IL-4, according to a classical method (10). As detected by RT-qPCR, the expression levels of arginase-1 and TGF-β1 in the eIF6+/+ and eIF6+/− mice significantly increased following IL-4 stimulation (P<0.05; Fig. 2).
Gene microarray analysis
A total of 1,716 differentially expressed genes were screened out of the gene expression profiles of the M2 macrophages from the eIF6+/+ and eIF6+/− mice. Compared with the eIF6+/+ mice, 851 genes in the macrophages of the eIF6+/− mice were downregulated and 865 were upregulated (Fig. 3). The expressions levels of fibrosis-associated genes, VEGF (eIF6+/+/eIF6+/−= 0.75; P<0.05) and TIMP-2 (eIF6+/+/eIF6+/−=0.65; P<0.05) in the M2 macrophages of the eIF6+/+ mice were significantly downregulated, whereas the expression of MMP-2 was significantly upregulated (eIF6+/+/eIF6+/−=1.3; P<0.05). However, the expression levels of other cytokines associated with wound healing and fibrosis, including epidermal growth factor (EGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), were similar (data not shown).
Validation of gene microarray results using RT-qPCR
Compared with the eIF6+/− mice, the M2 macrophages of the eIF6+/+ mice expressed significantly lower levels of VEGF and TIMP-2 (P<0.05), and a significantly higher level of MMP-2 (P<0.05; Fig. 4), consistent with the results of the gene microarray analysis. No significant inter-group differences were identified between the expression levels of EGF, TGF-β1, FGF or PDGF (data not shown).
Validation using ELISA
The supernatants of the M2 macrophages from the eIF6+/+ and eIF6+/− groups were collected, respectively, for analysis using ELISA. Compared with the eIF6+/− mice, the M2 macrophages from the eIF6+/+ mice expressed significantly lower levels of VEGF and TIMP-2 (P<0.05), whereas the level of MMP-2 was significantly higher in the eIF6+/+ mice (P<0.05; Fig. 4). These results were also in accordance with the results of the gene microarray analysis.
Discussion
eIF6 is a fibrosis-inhibitory gene, however, its mechanism of action remains to be fully elucidated. Macrophages are crucial in wound healing and fibrosis, thereforem, the aim of the present study was to investigate the effects of eIF6 on macrophages.
As multifunctional adaptive cells, macrophages can activate fibroblasts by secreting large quantities of cytokines, and are involved in ECM metabolism by regulating the MMP-2/TIMP-2 balance (11). Under in vitro culture conditions, M2 macrophages can be produced by induction with T helper 2 cytokines, including IL-4 and IL-13 (3,4). Different types of macrophages are now predominantly identified based on differences between membrane protein expression levels. For example, F4/80+CD11c−CD206+ macrophages are M2 macrophages (12,13). With the ability to express arginase-1, M2 macrophages are involved in the metabolism of ECM and mediate its damage repair function. In addition, they can secrete anti-inflammatory factors, including IL-10 and TGF-β1 (14). In the present study, macrophages were isolated from the peritoneal cavity and induced into M2 macrophages by being stimulated with 40 ng/ml IL-4 for 24 h. Flow cytometric and RT-qPCR analyses showed that the expression levels of CD206, arginase-1 and TGF-β1 were identical to those in the M2 macrophages obtained from wounds (4).
Granulation tissues, which are predominantly comprised of fibroblasts, inflammatory cells, new blood vessels and ECM (15), initially fill the wound defect and promote healing, and finally transform into scar tissue. Therefore, the quantity of granulation tissue produced determines the severity of scarring (16). In particular, angiogenesis predominantly controls the formation of granulation tissues (17). Macrophages secrete VEGF (18), which facilitates the migration and proliferation of vascular endothelial cells, as well as accelerating wound angiogenesis (19,20). In hypertrophic scars, numerous microvessels form, and the expression of VEGF markedly increases (21). In addition, inhibiting VEGF prevents the production of TGF-β1 via the PI3K-Akt signaling pathway, thereby suppressing pulmonary fibrosis (22). Accordingly, VEGF dominates in cicatrization and fibrosis. The expression of VEGF was significantly lower in the M2 macrophages from the eIF6+/+ mice, compared with those of the eIF6+/− mice, indicating that eIF6 decreased angiogenesis and affected scar tissue formation by inhibiting the production of VEGF.
Scars are also typified by considerable ECM deposition, and its metabolism is regulated by MMPs. MMPs are proteins, which are able to degrade ECM, of which MMP-2 (also known as gelatinase) is involved in ECM remodeling (23) by hydrolyzing type I/IV/V collagens, fibronectin and laminin (24,25). As specific inhibitory factors of MMPs, TIMPs form MMP-TIMP complexes by binding to the zinc active center, thus inhibiting the binding between MMPs and substrates. Therefore, the balance between MMPs and TIMPs has a marked effect on tissue repair and the growth of new epidermal cells. In the present study, compared with the eIF6+/− mice, the expression of MMP-2 in the M2 macrophages of the eIF6+/+ mice was increased, however, the expression of TIMP-2 was decreased, suggesting that eIF6 regulated the balance between MMP-2 and TIMP-2, accelerated ECM metabolism, reduced its deposition and eventually mitigated fibrosis.
In conclusion, the present study demonstrated that eIF6 relieved fibrosis/cicatrization by inhibiting the generation of VEGF to prevent the overgrowth of blood vessels and production of granulation tissues, and by regulating the MMP-2/TIMP-2 balance to promote ECM degradation and decrease its deposition. Therefore, eIF6 is a novel fibrosis-inhibitory gene, the functional analysis of which may lead to improvements in alleviating scarring.