The Effect Of Eukaryotic Initiation Factor 6 (eIF6) On Fibrosis Genes Of M2 Macrophage

Hypertrophic scar formation is a natural and inevitable physiological process in wound healing, but it also has some bad influence on the life of patients. Hypertrophic scar can interfere with the appearance and function of limbs and trunk, even cause serious psychological barrier. Even with the increasing knowledge of wound healing, hypertrophic scar is still difficult to prevent and treat. Therefore, it has been a big challenge for clinicians to effectively control the hypertrophic scar formationMacrophages play an important role in the process of wound healing and tissue fibrosis. It can engulf pathogens, secrete a diverse set of inflammatory cytokines and growth factors to suppress inflammation, removal necrotic tissue and promote wound healing. Recent in vivo studies on macrophages freshly isolated from human and murine acute wounds by enzymatic digestion demonstrate that wound macrophages adopt a diversity of phenotypes in response to different environmental cues during the tissue repair progression.Macrophages could displayed both M1 and M2 activation markers throughout wound healing, M1 macrophages prevail in the early inflammatory phases and secrete various inflammatory cytokines, while M2 macrophages increased in late inflammatory and secrete various growth factors.Eukaryotic initiation factor 6 (eIF6) plays a critical role in ribosome biogenesis and protein translation during the initial stage. eIF6 can combine with 60S ribosomal repressing 80S ribosomal formation, which is modulated by the RACK1-PKC complex.In the initiation stage of translation, PKC is recruited to the 40S subunit by RACK1 and phosphorylates eIF6 on Ser 235, which lead to eIF6 release from 60S and activates protein translation. Our previous study had found that eIF6 can act as a inhibitory factor of fibrosis in wound healing. The granulation tissue and the collagen content in eIF6+/- mice was significantly increased compared to eIF6 wild type mice in a mouse linear full-thickness incision wound model. Meanwhile, eIF6 overexpression reduced the collagen deposition compared to the control in a hepatic fibrosis model.Taken together, we hypothesis that eIF6 might be involved in the function of macrophages to regulate the wound healing. Here we screened for M2 macrophages genes that were differentially expressed in eIF6 wild type and eIF6+/- mice using gene microarrays, and then identified the conspicuously alterant genes among fibrosis.Methods1. Macrophages and M2 macrophages culture. The macrophages were isolated purified from the enterocoelia in both eIF6 wild type mice and eIF6+/- mice. Then the M2 macrophages were harvested after stimulation by IL-4 for 24 hours.2. M2 macrophages phenotype and function identification. The surface markers of M2 macrophages were identified by flow cytometry. The arginase-1 and TGF mRNA expressions were detected by RT-RCR.3. Gene microarrays. The M2 macrophages RNA from eIF6 wild type and eIF6+/- mice was extracted and detected by Invitrogen (Shanghai, China) for gene microarray analysis. The gene microarray was Affymetrix GeneChip Mouse Gene 1.0 ST Array, which was performed in three independent replicates.4. Gene verification. The M2 macrophages mRNA was harvested from eIF6 wild type and eIF6+/- mice. The RNA and protein expression was detected by RT-PCR and ELISA.Results1. The cells from abdominal cavity lavage fluid were cultured for 24 hours. The macrophage markers such as F4/80 and CD11b were positively expressed, and the purity of double positive cells reached to 94.4%.2. After activating by IL-4 for 24 hours, the CD2O6 expression was significantly increased while CD11c was negative by Flow cytometry analysis. 3. After activating by IL-4 for 24 hours, the mRNA levels of arginase 1 and TGF-β1 was significantly increased compared to the non-IL-4 treated groups by RT-PCR (P<0.05). 4. M2 macrophages genes from eIF6 wild type and eIF6+/- mice were screened using gene microarrays, and then identified the differentially expressed genes associated with fibrosis. Among these genes, VEGF (ratio 1.33, p< 0.05) and TIMP2 (ratio 1.54, p<0.05) were increased conspicuously in eIF6+/- mice. MMP2 (ratio 0.70, p<0.05) was decreased significantly in eIF6+/- mice.5. The VEGF and TIMP2 mRNA expression in eIF6+/- mice was up-regulated (P<0.05), while the MMP2 mRNA was down-regulated in eIF6+/- mice compared to eIF6 wild type by RT-PCR (P<0.01). These results were consistant with gene microarrays.6. The VEGF and TIMP2 protein levels in eIF6+/- mice were up-regulated (P< 0.05), while the MMP2 protein level was down-regulated in eIF6+/- mice compared to eIF6 wild type by ELISA (P<0.01). These results were consistant with gene microarrays.ConclusionM2 macrphages can secrect more VEGF when eIF6 knocking down, enhances angiogenesis and granulation tissue formation, and also regulate the degrees of fibrosis (scar formation) through affecting the extracellular matrix degradation and deposition by regulating the balance of MMP2/TIMP2.