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Effects of the Eukaryotic Initiation Factor 6 Gene on Expression Levels of Inflammatory Mediators in M2 Macrophages During Scar Repair

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Effects of the Eukaryotic Initiation Factor 6 Gene on Expression Levels of Inflammatory Mediators in M2 Macrophages During Scar Repair MOLECULAR MEDICINE REPORTS 14: 819-824, 2016 Effects of the eukaryotic initiation factor 6 gene on expression levels of inflammatory mediators in M2 macrophages during scar repair YONG-QIANG BAI*, JIAN-KE FENG*, QING-FU ZHANG, HONG-ZHI WU, YA-RU DU and WEI WEI Department of Burns and Plastic Surgery, The First Hospital of Hebei Medical University, Shijiazhuang, Hebei 050031, P.R. China Received April 16, 2015; Accepted March 14, 2016 DOI: 10.3892/mmr.2016.5294 Abstract. The aim of the present study was to evaluate the and fibrosis (1). Lucas et al (2) found that eliminating macro- effects of the eukaryotic initiation factor 6 (eIF6) gene on the phages delayed wound healing by decreasing granulation secretion of M2 macrophage fibrosis‑associated factors and tissue growth and inhibiting re‑epithelialization. By isolating the expression levels of key proteases during scar repair. Male and culturing macrophages from wounds, Sindrilaru et al (3) eIF6 wild-type (eIF6+/+) and knockout (eIF6+/-) C57BL/6 mice observed that different phenotypes were generated in response were intraperitoneally lavaged to obtain macrophages, which to various microenvironmental stimuli. Macrophages continu- were induced to the M2 type using interleukin-4. Differences ously express M1 (classic activation) and M2 (alternative between the gene expression profiles of these macrophages activation) surface markers throughout wound healing, with the were compared with gene microarrays, and the results were M1 and M2 types dominating at the beginning of the inflam- validated using reverse transcription-quantitative polymerase matory response and at the repair phase, respectively (4,5). As chain reaction analysis and ELISA. Compared with the eIF6+/- profibrotic cells, M2 macrophages secrete cytokines, which mice, the mRNA and protein expression levels of vascular activate the transformation of fibroblasts into myofibroblasts, endothelial growth factor (VEGF) and tissue inhibitor of thus promoting cicatrization by leading to the overgrowth metalloproteinase-2 (TIMP-2) in the M2 macrophages of of granulation tissues and deposition of extracellular matrix the eIF6+/+ mice were significantly downregulated (P<0.05), (ECM) (6). whereas the mRNA and protein expression levels of matrix Eukaryotic initiation factor 6 (eIF6), which regulates the metalloproteinase‑2 (MMP‑2) were significantly upregulated initial translation of eukaryotes, binds to large ribosomal (P<0.05). Therefore, the results indicated that eIF6 alleviated subunit 60S in cell nuclei to inhibit the formation of 80S ribo- cicatrization, possibly by inhibiting the generation of VEGF, in somes (7). At the beginning of protein translation, eIF6 is order to prevent overgrowth of blood vessels and granulation depolymerized from the 60S subunit through phosphorylation tissues, and to regulate the MMP-2/TIMP‑2 ratio to balance under the action of casein kinase 1-protein kinase C, which the degradation and deposition of the extracellular matrix. facilitates the binding between 40S and 60S subunits to form the 80S subunit, thereby initiating the translation process (8). Introduction Our previous study compared the wound healing outcomes of eIF6 wild-type (eIF6+/+) and knockout (IF6+/-) mice, and found Cicatrization, or fibrosis, is a normal, inevitable physiological that the latter had significantly more granulation tissue and response, which occurs during natural post‑burn healing, collagen, indicating that eIF6 inhibited skin fibrosis (unpub- however, it is clinically challenging due to the severe effect on lished data). However, the mechanism by which eIF6 affects quality of life. Macrophages are important in wound healing macrophages, wound healing and cicatrization remains to be RETRACTEDelucidated. 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, Correspondence to: Dr Wei Wei, Department of Burns and and a novel therapy strategy for mitigating scar repair and Plastic Surgery, The First Hospital of Hebei Medical University, 89 Donggang Road, Shijiazhuang, Hebei 050031, P.R. China fibrosis in clinical practice. E‑mail: [email protected] Materials and methods *Contributed equally Experimental animals and reagents. A total of 24 male eIF6 Key words: eukaryotic initiation factor 6, macrophage, fibrosis wild -type (eIF6+/+) and knockout (eIF6+/-) C57BL/6 mice (80 -1 weeks old; 15-20 g; Beijing HFK Bioscience Co., Ltd., Beijing, China) were used in the present study. The mice were 820 BAI et al: EFFECTS OF THE EUKARYOTIC INITIATION FACTOR 6 GENE IN SCARRING maintained in a temperature (18‑22˚C) and humidity (50‑60%) 1 ml of PBS to remove free antibodies, and resuspended in controlled environment with ad libitum access to food and 0.3 ml PBS. Finally, the expression levels of F4/80, CD11c and water. eIF6+/+ and eIF6+/- mice were maintained in 12 h/12 h CD206 on the cell surface were detected using flow cytometry. and 22 h/22 h light/dark cycles, respectively. Culture medium comprising RPMI 1640 and fetal bovine serum was purchased Gene microarray analysis. The RNAs of the M2 macrophages from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, from the eIF6+/+ and eIF6+/- mice were extracted and subjected USA). Recombinant mouse interleukin‑4 (IL‑4) was purchased to gene microarray analysis in triplicate. The detailed from Sigma-Aldrich (St. Louis, MO, USA). The following information is available at http://www.affymetrix.com. monoclonal mouse antibodies were purchased from Abcam (Cambridge, MA, USA): Anti‑F4/80-peridinin chlorophyll Reverse transcription-quantitative polymerase chain reaction protein complex (PerCP; 1:1,200 dilution; cat. no. ab111101), (RT -qPCR) analysis. Total RNA (1 g) was extracted from the anti‑CD11b‑fluorescein isothiocyanate (FITC; 1:900 dilution; macrophages using an RNeasy Mini kit and transcribed into cat. no. ab24874), anti‑Cd11c‑phycoerythrin (PE; 1:1,000 cDNA. Then, qPCR was performed in a 10 µl reaction system dilution; cat. no. ab155349), anti‑CD206(MR)‑FITC (1:1,000 using a 7700 Real-time Fluorescence Quantitative PCR dilution; cat. no. ab64693) and anti‑CD16/32 (1:1,200 dilution; System (Applied Biosystems; Thermo Fisher Scientific, Inc.) to cat. no. ab25235). The RNeasy Mini kit (cat. no. 2183018A) detect the expression levels of transforming growth factor-β1 was provided by Life Technologies; Thermo Fisher Scientific, (TGF-β1), arginase-1, matrix metalloproteinase-2 (MMP-2), Inc. The AMV3.0 Reverse Transcription kit was purchased tissue inhibitor of metalloproteinase‑2 (TIMP‑2) and vascular from Takara, Bio Inc. (Otsu, Japan; cat. no. RR019A). Gene endothelial growth factor (VEGF). The total reaction volume microarrays (Affymetrix GeneChip Mouse Gene1.0 ST Array) for PCR (25 µl) contained 12.5 µl 2X Premix Ex Taq, 1 µl were purchased from Affymetrix (Santa Clara, CA, USA). upstream and downstream primers, 1 µl cDNA and 9.5 µl The study was approved by the ethics committee of The First ddH2O. The PCR thermal cycling conditions were as follows: Hospital of Hebei Medical University (Shizjiazhuang, China). 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 Culture of peritoneal macrophages. The mice were sacrificed by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) and by cervical dislocation and the peritoneal cavity of each was the sequences were as follows: GAPDH, sense 3'- GGG GAA opened following sterilization with 75% ethanol and repeat- GGT GAA GGT CGG AGTC-5' and antisense 3'-GTC CTC CTG edly lavaged with 10 ml pre-cooled RPMI 1640 medium. The GAA GAT GGT GATG-5'; TGF-β1, sense 3'- TGG AAA CCC lavage fluid was collected into sterile centrifuge tubes placed ACA AA ACG A TCT ATGA-5' and antisense 3'- TGG AAA CCC on ice, and centrifuged at 4˚C and 12,000 x g for 5 min to ACA ACG AAA TCT ATGA-5'; arginase-1, sense 3'- CTC CAA obtain the cell precipitate, which was resuspended with 2 ml GCCAA A GTC CTT AGAG-5' and antisense 3'- GGA GCT GTC RPMI 1640 medium, containing 10% fetal bovine serum. ATT AGG GAC ATCA-5'; MMP-2, sense 3'- ACC TGA ACA Subsequently, the cells were inoculated at a density of 5x106 CTTTTG TC A GCTG-5' and antisense 3'- CTT CCG CAT GGT onto 6‑well plates and cultured in an incubator for 12 h at 37˚C CTC GATG-5'; TIMP-2, sense 3'-CAG ACC CCA TCA AGA in an incubator containing 5% CO2, of which those exhibiting GGA TTC-5' and anti -sense 3'-GGG GCC GTG TAG ATA AAC wall‑adherence were identified as macrophages. The stimu- TCG-5'; VEGF, sense 3'-CGC AGA CAG GGT TGC CATAC-5' lated group was treated with 40 ng/ml IL-4 for 24 h to acquire and antisense 3'- GGA GTG GGA TGG ATG ATG TCAG-5'. M2 macrophages, and an equal volume of phosphate‑buffered GAPDH was used as the internal reference, and the results saline (PBS) was added to the non-stimulated group. All other were expressed using the 2-ΔΔCq method (9). All experiments conditions were identical. were performed in triplicate. Detection of macrophage purity. The cultured macrophages ELISA. The supernatant obtained from the culture medium were digested with 0.5% trypsin (Sigma‑Aldrich), counted at of macrophages following stimulation with IL-4 for 24 h was the density of 5x105/tube, washed once with PBS, resuspended collected, and the expression levels of VEGF (cat. no. EK0506), in 100 µl PBS and incubated with 1 µl anti-CD16/32 for 10 min MMP -2 (cat. no. EK0511) and TIMP-2 (cat. no. EK0322) were at 37˚C to block the Fc receptor. The cells were then incubated detected using ELISA kits (Boster Biological Technology Co., with 1 µl anti-F4/80 -Percp and 1 µl anti‑CD11b‑FITC at room Ltd., Wuhan, China), in strict accordance with the manufac- temperature for 30 min, RETRACTEDwashed with 1 ml PBS to remove turer's protocol.
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