Elsevier

Cytokine

Volume 56, Issue 2, November 2011, Pages 256-264
Cytokine

Dysregulation of monocyte/macrophage phenotype in wounds of diabetic mice

https://doi.org/10.1016/j.cyto.2011.06.016Get rights and content

Abstract

The hypothesis of this study was that cells of the monocyte/macrophage lineage (Mo/Mp) exhibit an impaired transition from pro-inflammatory to pro-healing phenotypes in wounds of diabetic mice, which contributes to deficient healing. Mo/Mp isolated from excisional wounds in non-diabetic db/+ mice exhibited a pro-inflammatory phenotype on day 5 post-injury, with high level expression of the pro-inflammatory molecules interleukin-1β, matrix metalloprotease-9 and inducible nitric oxide synthase. Wound Mo/Mp exhibited a less inflammatory phenotype on day 10 post-injury, with decreased expression of the pro-inflammatory molecules and increased expression of the alternative activation markers CD206 and CD36. In contrast, in db/db mice, the pro-inflammatory phenotype persisted through day 10 post-injury and was associated with reduced expression of insulin-like growth factor-1, transforming growth factor-β1 and vascular endothelial growth factor. Reduced levels of these growth factors in wounds of db/db mice may have contributed to impaired wound closure, reduced granulation tissue formation, angiogenesis and collagen deposition. The persistent pro-inflammatory wound Mo/Mp phenotype in db/db mice may have resulted from elevated levels of pro-inflammatory interleukin-1β and interferon-γ and reduced levels of anti-inflammatory interleukin-10 in the wound environment. Our findings are consistent with the hypothesis that dysregulation of Mo/Mp phenotypes contributes to impaired healing of diabetic wounds.

Highlights

Diabetic wounds exhibit impaired healing and a persistent inflammatory response. ► Macrophages were isolated from excisional wounds of diabetic and non-diabetic mice. ► Macrophages in non-diabetic mice exhibit phenotypes associated with healing. ► Macrophages in diabetic mice exhibit a persistent pro-inflammatory phenotype. ► The persistent pro-inflammatory phenotype in diabetic mice may impede healing.

Introduction

Chronic wounds associated with diabetes, venous insufficiency and/or pressure represent a significant health problem with millions of patients afflicted and the associated treatment costing billions of dollars per year [1]. For diabetic patients, the lifetime incidence of a foot ulcer is up to 25% and up to 70% of these wounds remain unhealed after 20 weeks of standard treatment [2], [3], [4]. A common characteristic of these poorly healing wounds is a persistent inflammatory response, with prolonged accumulation of monocytes/macrophages (Mo/Mp) and elevated levels of pro-inflammatory cytokines [5], [6], [7], [8]. However, the role of Mo/Mp in the impaired healing of chronic wounds remains to be elucidated.

During normal wound healing, the classic functions of Mo/Mp are killing pathogens and clearing the wound of damaged tissue. Mo/Mp also directly promote healing, contributing to angiogenesis, collagen deposition and wound closure [9], [10], [11], [12]. In a recent study, we demonstrated that Mp depletion during wound healing in mice resulted in decreased wound levels of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β1 [12], suggesting that Mp are an important source of these growth factors during normal wound healing. However, during impaired healing of diabetic mice, wounds exhibit prolonged accumulation of Mo/Mp and pro-inflammatory cytokines and proteases, reduced levels of various growth factors, delayed closure, and reduced angiogenesis and matrix deposition, all of which mimic chronic wounds in humans [13], [14], [15]. One explanation for these findings is that the diabetic environment may perturb the function of Mo/Mp, which in turn could contribute to impaired healing. Indeed, Mp isolated from polyvinyl alcohol sponges implanted subcutaneously in db/db mice exhibit impaired phagocytosis of apoptotic cells [16].

Different microenvironments have the potential for inducing a wide range of Mo/Mp phenotypes or functions [17], [18], [19], [20]. Bacterial products and pro-inflammatory cytokines are known to induce the classical pro-inflammatory, pathogen killing Mo/Mp phenotype associated with production of high levels of inflammatory cytokines (e.g. interleukin (IL)-12, tumor necrosis factor (TNF)-α and IL-1β) and reactive oxygen and nitrogen species. Anti-inflammatory cytokines, glucocorticoids, and modulators of glucose and lipid metabolism induce a broad spectrum of “alternative” Mo/Mp phenotypes including those that exhibit non- or anti-inflammatory and pro-tissue repair functions, typically characterized by expression of mannose receptor (CD206), scavenger receptors (e.g. CD36) and growth factors such as TGF-β1 and insulin-like growth factor (IGF)-1. Although the term “alternative” activation was first ascribed to Mo/Mp stimulated with interleukin (IL)-4, the term has become a generic name used for any form of non-classical activation.

The vast majority of our knowledge about Mo/Mp phenotypes has been obtained from in vitro experiments in which phenotypes have been studied using stimulation with a single cytokine or other effector, or perhaps two of these effectors. In vivo, there is potential for a multitude of effectors to act on Mo/Mp and the phenotype expressed is likely due to the net effect of all of these signals. Mo/Mp along with other inflammatory cells, keratinocytes, fibroblasts and endothelial cells likely contribute to, and are likely influenced by, this complex molecular environment. Although Mo/Mp phenotypes have been described for cells isolated from sponges implanted subcutaneously in mice [16], [21], the phenotype of Mo/Mp isolated directly from skin wounds has been described only recently [22], but not for diabetic wounds. Thus, little is known about the role and regulation of different Mo/Mp phenotypes in skin wound healing, either during normal healing or in diabetic wounds. Our hypothesis is that, during normal skin healing in non-diabetic mice, wound Mo/Mp exhibit a pro-inflammatory phenotype early following injury and then express a pro-healing phenotype as healing progresses. We also hypothesize that this phenotypic transition is impaired in diabetic db/db mice, which may contribute to deficient healing.

Section snippets

Animals

Diabetic db/db mice and non-diabetic db/+ controls on a C57Bl/6 background were obtained from Jackson Laboratories (Bar Harbor, ME). Experiments were performed on 12–16 week-old mice. All experimental procedures were approved by the Animal Care Committee at the University of Illinois at Chicago.

Excisional wounding

Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) and their dorsum shaved and cleaned with betadine and then alcohol swab. Four 8 mm excisional wounds

Wound Mo/Mp differentiation

Although Mo/Mp play important roles in skin wound healing [9], [10], [11], [12], the phenotype(s) and regulation of skin wound Mo/Mp have not been elucidated. Thus, we isolated CD11b+/Ly6G− cells from excisional wounds to assess cells of the Mo/Mp lineage. We first measured expression of the Mo marker Ly6C and the Mp marker F4/80 – Ly6C expression decreases and F4/80 expression increases during Mo/Mp maturation [24], [25]. In CD11b+/Ly6G− wound cells isolated from db/+ mice, Ly6C mRNA

Discussion

Although Mp are required for normal wound healing, dysregulation of Mp function could contribute to impaired wound healing with diabetes [5], [6], [7], [8], [13], [14], [15]. The major finding of this study is that wound Mo/Mp of diabetic mice exhibit an impaired transition from pro-inflammatory to pro-healing phenotypes which may contribute to the pro-inflammatory wound environment and poor healing response. Wound Mo/Mp isolated from non-diabetic db/+ mice exhibited a pro-inflammatory

Acknowledgments

The authors thank Dr. Luisa DiPietro, University of Illinois at Chicago, for critical comments on a previous draft of this manuscript.

References (40)

  • K. Woo et al.

    The edge effect: current therapeutic options to advance the wound edge

    Adv Skin Wound Care

    (2007)
  • B.A. Mast et al.

    Interactions of cytokines, growth factors, and proteases in acute and chronic wounds

    Wound Repair Regen

    (1996)
  • K. Rosner et al.

    Immunohistochemical characterization of the cutaneous cellular infiltrate in different areas of chronic leg ulcers

    Apmis

    (1995)
  • N.J. Trengove et al.

    Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers

    Wound Repair Regen

    (2000)
  • S.J. Leibovich et al.

    The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum

    Am J Pathol

    (1975)
  • T. Lucas et al.

    Differential roles of macrophages in diverse phases of skin repair

    J Immunol

    (2010)
  • R. Blakytny et al.

    The molecular biology of chronic wounds and delayed healing in diabetes

    Diabet Med

    (2006)
  • I. Goren et al.

    Leptin and wound inflammation in diabetic ob/ob mice: differential regulation of neutrophil and macrophage influx and a potential role for the scab as a sink for inflammatory cells and mediators

    Diabetes

    (2003)
  • D.G. Greenhalgh et al.

    PDGF and FGF stimulate wound healing in the genetically diabetic mouse

    Am J Pathol

    (1990)
  • S. Khanna et al.

    Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice

    PLoS One

    (2010)
  • Cited by (0)

    View full text