Highlights:
After thermal injury, a polarized TH2 environment favors the subsequent development of increased TH3 cells (a different lineage from naturally arising CD25+ CD4+ Treg cells) and fibrocytes that can induce fibrosis in a paracrine fashion.
Evidence suggests that monocytes and other inflammatory cells may also contribute to the development of dermal fibrosis.
In animal models of trauma and fibrosis as well as humans with acute burn injury, evidence for reduced IL-2 and IFN-γ production and increased TH2 cytokines (IL-4, IL- 5, IL-10, IL-13) is emerging.103–105 In burn patients with HTS, a deficiency of circulating IFN-γ-producing lymphocytes exists very early after injury. Within 3 months postburn, increased numbers of IL-4-containing lymphocytes develop, which persist for up to 1 year after injury—consistent with a polarized TH2 response.104 Significant decreases in IL-10 within the first 2 months postinjury persist for 1 year, whereas IL-12 levels were significantly elevated and inversely related to IL-10.103 IFN-γ mRNA was not detected in PBMCs and in HTS tissues until 6 months postinjury. IL-4 was undetected in normal controls, but was increased in HTS patients in PBMCs within 2 months postinjury, as well as in HTS tissues (Fig. 6-5).103 CD4+ TGF-β+ lymphocytes are present in increased frequency in the circulating immune cells of burn patients as compared to normal control individuals.105 These cells secrete increased levels of TGF-β, which promotes proliferation of dermal fibroblasts as well as α-SMA and wound contraction. The development of CD4+ TGF-β+ cells may contribute to the suppression of TH1 immunity similar to trauma patients where increased T-regulatory CD4+ CD25+ cells, which produce TGF-β and other cytokines, have been found systemically.106,107 Pilling et al. have described that TH2 cytokines (IL-4, IL-13) promote, whereas TH1 cytokines (IFN-γ, IL-12) inhibit, fibrocyte differentiation in fibrosis.107
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FIGURE 6-5 Time course of IL-10 and IL-12 from peripheral blood mononuclear cells (PBMCs) isolated from burn patients. The percentages of IL-4 or IFN-positive cells in burn patient PBMCs determined by flow cytometry. (From Medina A, Ghahary A. Reprogrammed fibrocytes induce a mixed TH1/TH2 cytokine response of naïve CD4(+) T cells. Mol Cell Biochem. 2011 346(1/2):89–94.)
In liver and pulmonary fibrosis, macrophage subsets have been identified as key producers of TGF-β1 via TLR4 that promotes fibrosis. In addition to inducing fibrosis, TGF-β1 produced by regulatory T cells may suppress inflammation.108,109 Like lymphocytes, macrophages respond to TH2 cytokines where numerous studies have shown that macrophages exposed to IL-4 develop an alternate activation state termed “alternatively activated macrophages” or M2 macrophages, and their role may be equal to if not more important than the type of CD4+ T-helper cell response in many different types of fibrosis.109,110 Using an animal model of dermal fibrosis in human skin following transplantation into immunodeficient mice, animals which lack T cells, natural killer (NK) cells, and NK-T cells still develop significant fibrosis. This suggests that fibrosis can develop in the absence of lymphocytes and helps confirm an important role for other immune cells in the development of dermal fibrosis.111 For this reason, emphasis has shifted to the importance of the role of monocytes/macrophages in HTS and other forms of FPD.
M1/M2 Macrophages
Highlights:
M1 macrophages are associated with normal wound healing and signal through the NF-κB/STAT1 pathway.
M2 macrophages promote scar formation via the STAT3/STAT6 signaling pathway (Fig. 6-6).112
Since the initial discovery of macrophages by Metchnikoff in 1884, these mononuclear cells have been considered to play a vital role in the wound healing process. When they are ablated immunologically, studies have shown significant impairment in wound healing associated with a decreased number of macrophages at the injured site.113,114 Conversely, increased numbers of activated polarized macrophages can lead to disordered wound healing, eventuating in dermal fibrosis.115 Macrophages appear to differentiate into multiple phenotypes, but principally into classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages) that appear to have distinct opposite functions in the wound healing process.115 M1 macrophages induce nitric oxide synthase (NOS), which increases MMP-1 production and promotes ECM degradation, whereas M2 macrophages secrete large amounts of TGF-β1, which can stimulate myofibroblast differentiation and promote ECM deposition. It is also hypothesized that prolonged inflammation during wound healing attracts increased numbers of macrophages, which will initially be predominantly the proinflammatory M1 phenotype before a switch to the more profibrotic M2 phenotype occurs to counter the intense proinflammatory stimuli in the microenvironment.116,117 With the shift from M1 macrophages, arginine is metabolized to nitric oxide and citrulline via NOS as a part of the inflammatory process in the healing wound. With the resolution of inflammation, arginine is metabolized by arginase I (Arg- 1), leading to ornithine and polyamines modulated by M2 macrophages and leading to increased proline and collagen synthesis as well as other polyamines necessary for cell proliferation.118 Growing evidence suggests that M2 macrophages are not a uniform population but can be further subdivided into M2a, M2b, and M2c subsets.119 M2a macrophages are induced by IL-4 and IL-13, which are involved in an antiparasitic immune response and are considered to be profibrotic; M2b macrophages are induced by IL-1β, LPS, and immune complexes, whereas M2c macrophages are induced by IL- 10, TGF-β, and glucocorticoids.120 The fourth subtype of macrophages differentiates from an M1 phenotype into an angiogenic M2-like phenotype, which has been termed M2d by Leibovich et al.121,122
monocytes are recruited into the injured site via the SDF-1/CXCR4 signaling pathway and differentiate into polarized macrophages. The polarized M1 and M2 macrophages then exert their functions via various signaling pathways involved in wound healing and HTS formation. (From Zhu Z, Ding J, Ma Z, et al. The natural behavior of mononuclear phagocytes in HTS formation. Wound Repair Regen. 2016. 24(1):14–25. doi:10.1111/wrr.12378.)
Using an animal model of human skin transplanted to immunodeficient mice, Zhu et al.123 have demonstrated the development of contracted and thickened scars grossly. These resembled human HTS tissue histopathologically based on enhanced thickness, fibrotic orientation of collagen bundles, increased collagen level, and infiltration of myofibroblasts. Circulating monocytes decreased at 1 week after transplantation and returned to normal levels in the following 8 weeks. In the dermal tissues, F4/80+NOS2+ M1-like macrophages were found predominantly at 1 week after grafting, whereas F4/80+Arg-1+ M2-like macrophages were abundant at 3 weeks postgrafting (Fig. 6-7), coincident with the development of fibrosis in the human skin tissues. This understanding of the natural behavior of mononuclear phagocytes in vivo in the dermal mouse model of fibrosis provides evidence for the role of M2 macrophages in fibrosis of human skin, and suggests that macrophage depletion in the subacute phases of wound healing might reduce or prevent HTS formation.124
With the above discussion in mind, it is hypothesized that deep skin burn injury activates fibroblasts in the deep dermis via PAMPs like LPS and DAMPs like biglycan (and others) to stimulate the TLRs/NF-κB pathway in fibroblasts. This in turn releases chemokines and growth factors including TGF-β that recruit bone marrow–derived progenitor cells (such as M2 macrophages, fibrocytes, and TH2 cells) to further activate fibroblasts to produce excessive ECM proteins and ultimately to the development of HTS (Fig. 6-8).54
FIGURE 6-7 M1- and M2-like macrophages in xenografts. Human skin grafts were transplanted to the backs of nude mice. F4/80+NOS2+ M1-like macrophages were found predominantly at 1 week, whereas F4/80+Arg-1+ M2-like macrophages were abundant at 3 weeks postgrafting in the xenografts.