Mechanism Study Of The Regulatory Role Of Macrophages In Skin Damage Repair

Background: As the largest external organ of human and animal organisms,skin is extremely vulnerable to various types of damages.Consequently,skin damage repair(also known as wound repair)has been a frequent but meanwhile challenging topic in medical research.Wound repair is an important and complex process,involving four overlapping phases,which are hematopoietic,inflammatory,proliferative and remodeling phases.In addition to traditional skin grafting methods,new therapies employing tissue engineering skin,growth factors administration and cell and photobiomodulation treatments are increasingly used clinically during the process of skin damage healing.However,problems remain to be solved.These include high costs,rigorous requirement for storage,patient to patient variation post treatment and side-effects of potential oncogenicity.Besides,with the increasing demand from patients,it is imperative to optimize the existing therapies and develop new treatments.As an important member in the immune system,macrophages not only respond promptly to external invasion,but also take part in the repair and regeneration process of various tissues and organs.During the damage repair process of tissues,such as peripheral nerve,brain,kidney,muscle and bone,macrophages can,on the one hand,secrete growth factor and activate signaling pathways to promote the process.On the other hand,by playing a role in phagocytosis,macrophages can remove necrotic cells and prevent further damage.Lack of macrophages directly interferes with the formation of blastema during the regeneration of appendages of amphibians,thus impairs the entire repair process.Currently,in the skin tissues,whether macrophages play by a similar mechanism and the process of how they participate in wound healing are still unclear.Based on these research theories,using mice as the target animal,the aim of this study were firstly to observe the repairing stages post full-thichness dorsal skin excision and the distribution of macrophages in skin tissues,then to use macrophage-depleted models to assess the influence of healing process at the histological,gene and protein levels.Secondly,GM-CSF was injected subcutaneously to systematically stimulate the proliferation and differentiation of mononuclear-macrophage lineage,in which wound healing process was traced and changes in gene expression were determined.Finally,protein extracts from skin tissue of GM-CSF-treated mice were analyzed by LC-MS/MS.Protein molecules with significant changes in their expression during the early phases of the repair process were identified and annotated to their related biological process and signaling pathways.Results from this study will underpin the theoretical basis for elucidating the role of macrophages in the process of wound healing,and provide insights for developing new therapeutical treatments.Methods: This study was divided into four parts:1.Mice dorsal skin excision was performed,wounds were photographed and histologically stained at different time points.Repair rate,re-epithelialization level,macrophage distribution pattern and numbers were observed;2.Mice were treated with Liposome Clodronate(L-clod)to establish macrophage depletion mouse model and Liposome PBS(L-PBS)-treated mice were used as the control.Assays of histological,gene and protein determination were performed to evaluate the effects of macrophages on the skin damage repair process;3.Mice were injected with GM-CSF to stimulate the differentiation and proliferation of mononuclear-macrophage lineage.The number of macrophages in the circulation system was analyzed by flow cytometry and the effect of treatments on promoting skin wound healing process was evaluated.Genetic expression profiles of regeneration associated cytokines were assessed;4.Total protein was extracted in the skin tissues at 0,3 and 6 dpi in mice injected with GM-CSF and the equivalents in the control group.Differentially expressed proteins were identified by LC-MS/MS.The results were enriched and the main biological processes and signaling pathways associated were analyzed and further discussed.Result: 1.Following the skin excision damage,a large amount of granulation tissue appeared at 5 dpi.Re-epithelialization was completed at 10 dpi and by the end of 15 dpi,the granulation tissue diminished and the wound was completely healed.During this process,macrophages were constantly visible in the damaged skin tissue,and were evenly distributed therein.2.After the treatment of L-clod,the number of macrophages in the skin tissue was greatly reduced.The repair rate and re-epithelialization level of the experimental group was significantly lower than those of the control group at 6 and 9 dpi.Although the skin from both macrophage depletion and their control groups had no difference in collagen volume,the deposition speed of the experimental group was relatively slow and the collagen fiber rearrangement process was delayed comparing with that of the mice treated with L-PBS.q RT-PCR and ELISA analysis demonstrated that most of the inflammatory factors,cytokines and growth factors detected were down-regulated.3.After subcutaneous injection of GM-CSF,the number of monocytes-macrophage in peripheral blood significantly increased with a faster wound healing rate.Collagen volume in the neo-skin tissue from the experimental mice was relatively low to that of the control mice,indicating that scar formation was reduced after GM-CSF injection.In addition,regenerative genes associated with EMT,angiogenesis and ECM degradation were significantly up-regulated in the experimental group.4.The LC-MS/MS results showed that the expression of various proteins in GM-CSF-injected mice were up-regulated,and a large part of them were involved in the early repair process,including cell adhesion and chemotaxis,antigen processing and presentation,lysosomes,coagulation reactions and various cytokine signaling pathways.Besides,most of them had strong protein-protein interaction and functioned in the same networks.Conclusion: The results showed that macrophage was crucial to skin damage repair and had participated in and many pathways by up-regulating them in this process.Administration of GM-CSF had positive effect on stimulating the differentiation and proliferation of mononuclear-macrophage lineage in the peripheral blood circulation.This treatment augmented the expression level of various proteins related to the early stage of the wound healing process and thus regulated and activated signaling pathways related with inflammation,cell migration and proliferation.These biological processes were beneficial to promote wound healing,accelerated re-epithelialization and reduced scar formation.Those results preliminarily exemplified the positive influence of GM-CSF injection in promoting the skin damage repair process,which undoubtedly laid the foundation for future research directions by making it feasible to develop novel GM-CSF based therapies.