Polarized Macrophages in Hypertrophic Scar Formation

  • Author / Creator
    Zhu, Zhensen
  • Hypertrophic scars (HTS) are caused by trauma or burn injuries to the deep dermis and can cause cosmetic disfigurement and psychological issues. Our lab has established a human HTS-like nude mouse model, in which the grafted human skin develops red, raised, and firm scarring, resembling hypertrophic scars seen in humans. Macrophages play a key role in the wound healing process and can be divided into classically activated macrophages (M1) and alternatively activated macrophages (M2). Monocytes, M1 and M2 macrophages belong to the mononuclear phagocyte system (MPS). Studies suggest that M2 macrophages are pro-fibrotic and might contribute to HTS formation. In order to test that M2 macrophages contribute to HTS formation, three consecutive experiments were conducted. The first experiment was to test the effect of M2 macrophages on the fibrogenic activities of the cultured human dermal fibroblasts (HDF). In this study, resting state (M0), M1 and M2 macrophages differentiated from the human monocytic THP-1 cell line were used to co-culture with HDF for 48, 96 and 144 hours. The differentiation and polarization from THP-1 cells to M0, M1 and M2 macrophages were characterized by flow cytometry and cell cycle analysis. Cell sorting was performed to purify M0 and M2 macrophages. Cell proliferation, collagen synthesis, myofibroblast formation, gene expression of anti-fibrotic and pro-fibrotic factors, MMP-1 activity, and cytokine concentration were investigated. Results showed differentiation of M0 and polarization of M1 and M2 macrophages. M2 macrophages promoted the fibrogenic activities of co-cultured HDF by facilitating cell proliferation, increasing the collagen content, alpha-SMA expressing cells, expression of the pro-fibrotic genes and concentration of M2 macrophage related factors, as well as decreasing the expression of the anti-fibrotic genes and MMP-1 activity. These findings reinforce the pro-fibrotic role of M2 macrophages, suggesting therapeutic strategies in fibrotic diseases should target M2 macrophages in the future. The second experiment was to explore the natural behavior of MPS in the human HTS-like nude mouse model. Thirty athymic nude mice received human skin grafts (xenograft) and another thirty mice received mouse skin grafts (allograft) as controls. The grafted skin and blood were harvested at 1, 2, 3, 4 and 8 weeks. Wound area, thickness, collagen morphology and level, the cell number of myofibroblasts, M1 and M2 macrophages in the grafted skin were investigated. Xenografted mice developed contracted and thickened scars while the xenografted skin resembled human hypertrophic scar tissue based on enhanced thickness, fibrotic orientation of collagen bundles, increased collagen level and infiltration of myofibroblasts. In the xenografted skin, M1 macrophages were found predominantly at 1 to 2 weeks while M2 macrophages were abundant at 3 to 4 weeks post-grafting. This understanding of the natural behavior of mononuclear phagocytes in our mouse model provides evidence for the role of macrophages in human dermal fibrosis and suggests that macrophage depletion in the subacute phases of wound healing might reduce or prevent HTS formation. The third experiment was to study the effect of systemic macrophage depletion on scar formation at the subacute phase of wound healing. Thirty-six athymic nude mice that received human skin transplants were randomly divided into macrophage depletion group and control group. The former received intraperitoneal injections of clodronate liposomes while the controls received sterile saline injections on day 7, 10, 13 post-grafting. Wound area, scar thickness, collagen abundance and collagen bundle structure, mast cell infiltration, myofibroblast formation, M1 and M2 macrophages together with gene expression of M1 and M2 related factors in the grafted skin were investigated at 2, 4 and 8 weeks post-grafting. The skin grafts from the control group developed contracted, elevated and thickened scars while the grafted skin from the depletion group healed with significantly less contraction and elevation. Significant reductions in myofibroblast number, collagen synthesis and hypertrophic fiber morphology as well as mast cell infiltration were observed in the depletion group compared to the control group. Macrophage depletion significantly reduced M1 and M2 macrophage number in the depletion group 2 weeks post-grafting as compared to the control group. Systemic macrophage depletion at subacute phase of wound healing showed reduced scar formation. These findings provide evidence for the pro-fibrotic role of M2 macrophages in human dermal fibrosis as well as insight into the potential benefits of specifically depleting M2 macrophages in vivo.

  • Subjects / Keywords
  • Graduation date
    Fall 2016
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Specialization
    • Experimental Surgery
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Nedelec, Bernadette (Occupational Therapy)
    • Ding, Jie (Surgery)
    • Tredget, Edward E (Surgery)
    • Churchill, Thomas A (Surgery)
    • Lacy, Paige (Pulmonary Medicine)
    • Anderson, Colin (Surgery)
    • Agrawal, Babita (Surgery)